# Patent

## Title
Methods for detection of parvovirus B19

## Abstract

Nucleic acid molecules derived from sequences of novel human parvovirus B19 variant genomes are provided. Also provided are assays and kits comprising the nucleic acid molecules.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a continuation of U.S. patent application Ser. No. 12/523,309, filed on Jul. 17, 2009, which is a National Phase Entry under §371 of International Application No. PCT/US08/51083 filed Jan. 15, 2008, which claims the benefit of priority to U.S. Application No. 60/885,074 filed Jan. 16, 2007, and U.S. Application No. 60/942,762 filed Jun. 8, 2007, each of which is herein incorporated by reference in its entirety.


FIELD OF THE INVENTION
The present invention relates to human erythrovirus and includes methods and compositions useful for detection of a novel variant.
BACKGROUND OF THE INVENTION
Members of the Parvoviridae family of viruses are common animal and insect pathogens, which are further classified into the subfamily Parvoviridae based at least on the ability to infect vertebrate cells. Parvovirinae belonging to the genus Erythrovirus are known to infect humans and include, for example, the prototypical parvovirus B19 referred to as Au (genotype 1) as well as variants such as A6 (genotype 2), and V9 and D91.1 (genotype 3). They are non-enveloped viruses that comprise a single-stranded, linear DNA genome. For example, the prototypical human erythrovirus known as parvovirus B19-Au (See e.g., GenBank Accession Number: M13178) has a linear DNA genome of approximately 5.6 kilobases in length.
Discovered in 1975, parvovirus B19 was subsequently linked to an aplastic crisis in a patient with sickle-cell disease. The virus has since been shown to cause or be associated with a variety of conditions and diseases including erythma infectiosum (EI) (fifth disease of childhood), spontaneous abortion, and certain forms of acute arthritis.
Erythrovirus are ubiquitous and contagious. In the case of parvovirus B19, an estimated 60% of adults are seropositive. Children are particularly susceptible at the age when they begin to play together regularly and attend school, the peak season for infection being in the spring and early summer.
In addition to transmission through airborne infections and close contact, human erythrovirus can also be transmitted vertically from a pregnant woman to her fetus. For example, among pregnant women with active cases, about 30% of the fetuses will become infected with parvovirus B19. And, it is now well documented that parvovirus B19 can cause spontaneous abortion when infection occurs within the first six weeks after conception. Infection at this early stage causes massive abnormalities that are inconsistent with life.
Transmission of human erythrovirus also can occur via blood or plasma products of various kinds. For example, cases of symptomatic illness have been reported to be due to blood products prepared from parvovirus B19-containing plasma pools. Parvovirus B19 DNA has been detected in single donations, in plasma pools, and in plasma derivatives (e.g., clotting factors, albumin, antithrombin III, and immunoglobulins) produced by different processes. Parvovirus B19 transmission has also been found in patients treated with clotting factors, as shown by a higher seroprevalence in treated hemophiliacs, by the presence of parvovirus B19 DNA, and by active seroconversion. Unfortunately, the risk of human erythrovirus transmission by blood/plasma products is enhanced by the virus's resistance to effective inactivation methods such as heat and solvent-detergent treatments.
Therefore, health risks from exposure to human erythrovirus continue to exist, and identification and characterization of variants of the Erythrovirus genus will constitute an important step towards proper diagnosis and management of infection. Immunodiagnostic methods have been used to test blood, serum, or plasma that is potentially contaminated with human erythrovirus. But such immunodiagnostic methods have limitations including, for example, inability to effectively detect recent or current infections and/or inability to distinguish between the different erythrovirus genotypes. There is still a need, therefore, for identifying and characterizing human erythrovirus variants and developing sensitive and effective assays for detecting them and/or distinguishing from among them.
SUMMARY OF THE INVENTION
There is now provided an isolated nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO: 1, SEQ ID NO:128, SEQ ID NO:129, or a complement thereof.
In one aspect, the present invention provides nucleic acid molecules that are individually useful for detection of Parvoviridae represented by the nucleic acid sequence of SEQ ID NO:1 (i.e., partial genomic sequence of the novel variant named D11 disclosed herein), as well as those of known parvovirus B19 Au genotype 1, A6 genotype 2, V9 genotype 3, and D91.1 genotype 3. Accordingly, the invention includes an isolated nucleic acid molecule comprising a sequence consisting of at least 12, at least 15, or at least 20 contiguous nucleotides or a complement thereof, wherein the contiguous nucleotides are contained in a nucleotide sequence selected from the group consisting of:







(SEQ ID NO: 2)


5′-GATAACTGGTGGTGCTCT-3′;


 


(SEQ ID NO: 3)


5′-ACTTCTGACTGGGA-3′;


 


(SEQ ID NO: 4)


5′-GAATGTAACAAATTTGA-3′;


 


(SEQ ID NO: 5)


5′-TTATTTAATAATGT-3′;


 


(SEQ ID NO: 6)


5′-CTTGTAACTGAAA-3′;


 


(SEQ ID NO: 7)


5′-TTTAGAGATGGAGA-3′;


 


(SEQ ID NO: 8)


5′-TTAATGAAAAAAAT-3′;


 


(SEQ ID NO: 9)


5′-CCTTTAAATGTTGT-3′;


 


(SEQ ID NO: 10)


5′-CAGACTTTGAGCAGG-3′;


 


(SEQ ID NO: 11)


5′-TGGAATAATGAAAA-3′;


 


(SEQ ID NO: 12)


5′-TTTCCATTTAATGATGTAGC-3′;


 


(SEQ ID NO: 13)


5′-TTGGTGGTCTGGGATGA-3′;


 


(SEQ ID NO: 14)


5′-GAAGCTGCAAAAGCCATTTTAGG-3′;


 


(SEQ ID NO: 15)


5′-ACCAGGGTAGATCA-3′;


 


(SEQ ID NO: 16)


5′-ATAACCAGCAATGGTGACATTAC-3′;


 


(SEQ ID NO: 17)


5′-CATGCTAAAGCCTTAAA-3′;


 


(SEQ ID NO: 18)


5′-AGCCCTGACATGGG-3′;


 


(SEQ ID NO: 19)


5′-TGGTGTAATGCACAAAGCTGG-3′;


 


(SEQ ID NO: 20)


5′-CCACTATGAAAACTGGGCAATAAACTACAC-3′;


 


(SEQ ID NO: 21)


5′-TTTGATTTCCCTGGAAT-3′;


 


(SEQ ID NO: 22)


5′-AATGCAGATGCCCTCCACCCAGA-3′;


 


(SEQ ID NO: 23)


5′-CTCCAAACCACCCC-3′;


 


(SEQ ID NO: 24)


5′-TCAGCAGCAGTGGTGGTGAAAGCTCTGAAGAACTC-3′;


 


(SEQ ID NO: 25)


5′-CCAGGCGCCTGGAACA-3′;


 


(SEQ ID NO: 26)


5′-TGAAACCCCGCGCTCTAGTACGCC-3′;


 


(SEQ ID NO: 27)


5′-TCCCCGGGACCAGTTCAGGAGAATCATTTGTCGGAAGC-3′;


 


(SEQ ID NO: 28)


5′-CAGTTTCGTGAACTGTTAGT-3′;


 


(SEQ ID NO: 29)


5′-GCTTGGTATAATGGATGGAA-3′;


 


(SEQ ID NO: 30)


5′-AAATGTGCTTACCT-3′;


 


(SEQ ID NO: 31)


5′-TTTGTAGATTATGAGTAAA-3′;


 


(SEQ ID NO: 32)


5′-ATTTCTTTAGATAATCC-3′;


 


(SEQ ID NO: 33)


5′-TATATAGTCATCATTTTCA-3′;


 


(SEQ ID NO: 34)


5′-CATGGACAGTTATCTGACCACCCCCATGCCTTATCATCCAGTA-3′;


 


(SEQ ID NO: 35)


5′-CAGAACCTAGAGGAGAAAATGCAGTATTATCTA-3′;


 


(SEQ ID NO: 36)


5′-TGAAGACTTACACAAGCCTGGGCAAGTTAGC-3′;


 


(SEQ ID NO: 37)


5′-TACCCGGTACTAACTATGTTGGGCCTGGCAATGAG-3′;


 


(SEQ ID NO: 38)


5′-TACAAGCTGGGCC-3′;


 


(SEQ ID NO: 39)


5′-GACAGTGCTGCAAGGATTCATGACTTTAGGTATAGCCAA-3′;


 


(SEQ ID NO: 40)


5′-TTAAAAAATATAAAAAATGAAAC-3′;


 


(SEQ ID NO: 41)


5′-TACTTTACTTTAAAAGGTGCAGCTGCCCCTGTGGCCCATTTTCAAGG


 


AAGTTT-3′;


 


(SEQ ID NO: 42)


5′-TACAACGCCTCAGAAAAATACCC-3′;


 


(SEQ ID NO: 43)


5′-AGCATGACTTCAGTTAA-3′;


 


(SEQ ID NO: 44)


5′-TCTGCAGAAGCCAGCACTGGTGCAGG-3′;


 


(SEQ ID NO: 45)


5′-AAAAGCATGTGGAGTGA-3′;


 


(SEQ ID NO: 46)


5′-AGTAGCTGCCACAATGC-3′;


 


(SEQ ID NO: 47)


5′-TTAGATTTTAATGCTTT-3′;


 


(SEQ ID NO: 48)


5′-GATGCTTTAACTGT-3′;


 


(SEQ ID NO: 49)


5′-TATGCTTACTTAACAGTAGG-3′;


 


(SEQ ID NO: 50)


5′-AGTGAAGAATCAGC-3′;


 


(SEQ ID NO: 51)


5′-TTTTATGAAATGTACAA-3′;


 


(SEQ ID NO: 52)


5′-GCTGAAGACAAAGAGTATCA-3′;


 


(SEQ ID NO: 53)


5′-AATGAAAAAGAACA-3′;


 


(SEQ ID NO: 54)


5′-TGGAACAGAAGAGC-3′;


 


(SEQ ID NO: 55)


5′-CTTCACTATGAAAG-3′;


 


(SEQ ID NO: 56)


5′-CCTCAAATATTTTTAAAAATA-3′;


 


(SEQ ID NO: 57)


5′-CCTCAAATATTTTTAAAAATA-3′;


 


(SEQ ID NO: 58)


5′-CATTTACCATATGTACT-3′;


 


(SEQ ID NO: 59)


5′-TATGACCCCACAGCTACAGATGCAAA-3′;


and


 


(SEQ ID NO: 60)


5′-GGATATGAAAAGCCTGAAGAATTGTGGAC-3′.






In another aspect, the present invention provides nucleic acid molecules that are individually useful for specific detection of the Parvoviridae represented by the nucleic acid sequence of SEQ ID NO:1. Accordingly, the invention includes an isolated nucleic acid molecule comprising at least 12, at least 15, or at least 20 contiguous nucleotides of a nucleotide sequence or a complement thereof. The nucleotide sequence is selected from the group consisting of









(SEQ ID NO: 61)



5′-CACTTCTGACTGGGAACCATTAACTCATTCTAACAGACT-3′,



 



(SEQ ID NO: 62)



5′-ATGTAAAGCTTAAATTTTTACCAGGAATGACTACAAAAG-3′,



 



(SEQ ID NO: 63)



5′-AATATTTTAGAGATGGAGAACAATTTATAGAAAATTATT-3′,



 



(SEQ ID NO: 64)



5′-ATTTTAGAGATGGAGAACAATTTATAGAAAATTATTTAA-3′,



 



(SEQ ID NO: 65)



5′-TAACCAATATTGATGGGTACATAGATACCTGCATTTCTG-3′,



 



(SEQ ID NO: 66)



5′-ATGGGTACATAGATACCTGCATTTCTGCTTCTTTTAGAC-3′,



 



(SEQ ID NO: 67)



5′-TTAGACGGGGAGCCTTTCAGGCTAAAAAACCCCGCATTA-3′,



 



(SEQ ID NO: 68)



5′-GAACCAGGGGAATCTAGCGCTACAGGGGGAGATGTTGTG-3′,



 



(SEQ ID NO: 69)



5′-TGCCATTTGCTGGGAAGGGGACTAAAGCTGGAATAAAAT-3′,



 



(SEQ ID NO: 70)



5′-GGACTAAAGCTGGAATAAAATTTCAAACTATGGTAAATT-3′,



 



(SEQ ID NO: 71)



5′-TAAATTGGTTGTGTGAAAATAGGGTTTTTACAGAGGATA-3′,



 



(SEQ ID NO: 72)



5′-ATTGGTTGTGTGAAAATAGGGTTTTTACAGAGGATAAGT-3′,



 



(SEQ ID NO: 73)



5′-TTAACCAGTACACTTTACTTAGCAGTAGTCACAGTGGGA-3′,



 



(SEQ ID NO: 74)



5′-TAAAACTAGCTATTTATAAGGCTACCAATTTAGTGCCTA-3′,



 



(SEQ ID NO: 75)



5′-TAGCTATTTATAAGGCTACCAATTTAGTGCCTACAAGTA-3′,



 



(SEQ ID NO: 76)



5′-CTACCAATTTAGTGCCTACAAGTACATTTTTGTTACACA-3′,



 



(SEQ ID NO: 77)



5′-CAAGTACATTTTTGTTACACACAGACTTTGAGCAGGCTA-3′,



 



(SEQ ID NO: 78)



5′-CACACAGACTTTGAGCAGGCTAACTGTATTAAAGAAAAT-3′,



 



(SEQ ID NO: 79)



5′-GTGTCAAAATTATGACCCCTTGTTGGTGGGACAGCATGT-3′,



 



(SEQ ID NO: 80)



5′-GGATTGATAAAAAATGTGGCAAAAAAAATACACTGTGGT-3′,



 



(SEQ ID NO: 81)



5′-ATACACTGTGGTTTTATGGCCCACCAAGTACAGGAAAAA-3′,



 



(SEQ ID NO: 82)



5′-GTACAGGAAAAACAAATTTAGCAATGGCTATTGCTAAAA-3′,



 



(SEQ ID NO: 83)



5′-GCTTGGTGGTCTGGGATGAGGGTATTATTAAGTCTACTA-3′,



 



(SEQ ID NO: 84)



5′-GCTTACTTACAGAGGCTGACGTGCAGCAATGGCTTACAT-3′,



 



(SEQ ID NO: 85)



5′-CCCCGCGCTCTAGTACGCCAGTCCCCGGGACCAGTTCAG-3′,



 



(SEQ ID NO: 86)



5′-AGAATCATTTGTCGGAAGCTCAATTTCCTCCGAAGCTGT-3′,



 



(SEQ ID NO: 87)



5′-ATCATTTGTCGGAAGCTCAATTTCCTCCGAAGCTGTAGC-3′,



 



(SEQ ID NO: 88)



5′-AGCTCAATTTCCTCCGAAGCTGTAGCTGCATCGTGGGAA-3′,



 



(SEQ ID NO: 89)



5′-TGACTATGTATGGGATGGTATAAGGGGTTTACCTGTTTG-3′,



 



(SEQ ID NO: 90)



5′-TTAATAACAGTGGGGGAGGCTTGGGATTTTGTCCCCATT-3′,



 



(SEQ ID NO: 91)



5′-CAGTGGGGGAGGCTTGGGATTTTGTCCCCATTGCATTAA-3′,



 



(SEQ ID NO: 92)



5′-GCAAAAAATGTGCTTACCTATCTGGCTTGCAAAGTTTTG-3′,



 



(SEQ ID NO: 93)



5′-AATGTGCTTACCTATCTGGCTTGCAAAGTTTTGTAGATT-3′,



 



(SEQ ID NO: 94)



5′-TTTGTAGATTATGAGTAAAGAAATTGGTAAATGGTGGGA-3′,



 



(SEQ ID NO: 95)



5′-TTATGAGTAAAGAAATTGGTAAATGGTGGGAAAGTGATG-3′,



 



(SEQ ID NO: 96)



5′-CTTCTTTGTTTGACTTAGTGGCTCGTATTAAAAGTAACC-3′,



 



(SEQ ID NO: 97)



5′-ATGAAACTGGGTTTCAAGCTCAAGTAGTAAAAGACTACT-3′,



 



(SEQ ID NO: 98)



5′-TCCTGATGCTTTAACTGTTGCCATATCAGAAATTGCCAT-3′,



 



(SEQ ID NO: 99)



5′-TTGCCATATCAGAAATTGCCATTAAAGATGTTACAGACA-3′,



 



(SEQ ID NO: 100)



5′-TGCCATATCAGAAATTGCCATTAAAGATGTTACAGACAA-3′,



 



(SEQ ID NO: 101)



5′-AATACAAGTACCCATATGTATTAGGTCAAGGACAAGATA-3′,



 



(SEQ ID NO: 102)



5′-AAGATACCTTAGCCCCAGAGCTTCCAATTTGGGTGTACT-3′,



 



(SEQ ID NO: 103)



5′-CAGTAGGAGATGTAAACACGCAGGGAATTTCTGGGGACA-3′,



 



(SEQ ID NO: 104)



5′-AGAATCAGCGTTTTATGTCCTGGAACACAGCTCTTTTGA-3′,



 



(SEQ ID NO: 105)



5′-CTACTATGTCTTATAAGTTCCCTCCAGTGCCCCCAGAGA-3′,



 



(SEQ ID NO: 106)



5′-TCCCTCCAGTGCCCCCAGAGAATTTAGAAGGCTGTAGTC-3′,



 



(SEQ ID NO: 107)



5′-CCCGTTTAGGAGTCCCTGATACATTAGGAGGGGACCCCA-3′,



 



(SEQ ID NO: 108)



5′-AACACATGAAGACCACGCAGTTCAGCCACAAAATTTTAT-3′,



 



(SEQ ID NO: 109)



5′-ACGCAGTTCAGCCACAAAATTTTATGCCAGGGCCACTGG-3′,



 



(SEQ ID NO: 110)



5′-GGCCACTGGTAAACTCAGTTTCCACAAAGGAGGGAGACA-3′,



 



(SEQ ID NO: 111)



5′-AGGAGGGAGACAGTTCTAACACAGGAGCGGGAAAAGCCC-3′,



 



(SEQ ID NO: 112)



5′-GTCAAAGTACTAGAATATCATTACGCCCTGGTCCAGTGT-3′,



 



(SEQ ID NO: 113)



5′-GCCCTGGTCCAGTGTCTCAACCATATCACCACTGGGACA-3′,



 



(SEQ ID NO: 114)



5′-GTCCAGTGTCTCAACCATATCACCACTGGGACACAGATA-3′,



 



(SEQ ID NO: 115)



5′-CAGATAAATATGTAACAGGGATAAATGCCATTTCTCATG-3′,



 



(SEQ ID NO: 116)



5′-CTGAAGACAAAGAGTATCAACAGGGCGTGGGTAGGTTTC-3′,



 



(SEQ ID NO: 117)



5′-AAGACAAAGAGTATCAACAGGGCGTGGGTAGGTTTCCCA-3′,



 



(SEQ ID NO: 118)



5′-AGGGCGTGGGTAGGTTTCCCAATGAAAAAGAACAACTAA-3′,



 



(SEQ ID NO: 119)



5′-AACAGTTACAGGGTTTAAATATACACACATATTTTCCCA-3′,



 



(SEQ ID NO: 120)



5′-GTTTAAATATACACACATATTTTCCCAATAAAGGTACCC-3′,



 



(SEQ ID NO: 121)



5′-TACCAAATTTAGATGACAGCTTTAAAACTCAGTTTGCAG-3′,



 



(SEQ ID NO: 122)



5′-AGCTTTAGGAGGTTGGGGACTACATCAGCCACCCCCTCA-3′,



 



(SEQ ID NO: 123)



5′-GGCCAATTGGGGGTATTAAGTCAATGGGAATAACAACAT-3′,



 



(SEQ ID NO: 124)



5′-TTAAGTCAATGGGAATAACAACATTAGTTCAATATGCTG-3′,



 



(SEQ ID NO: 125)



5′-TAGTTCAATATGCTGTGGGTATTATGACAGTAACTATGA-3′,



 



(SEQ ID NO: 126)



5′-TAACTATGACATTTAAATTAGGGCCTCGCAAAGCTACAG-3′,



and



 



(SEQ ID NO: 127)



5′-ACCCTCCTCACGCAGCAGGCCATTTACCATATGTACTAT-3′.






In other aspects, the present invention provides an isolated nucleic acid molecule consisting of a nucleotide sequence or a complement thereof, wherein the nucleotide sequence is selected from the group consisting of:









(SEQ ID NO: 136)



5′-TGAAACCCCGCGCTCTA-3′;



 



(SEQ ID NO: 137)



5′-AACTAACAGTTCACGAAACTG-3′;



 



(SEQ ID NO: 138)



5′-TCCCCGGGACCAGTTCAGGAGAA-3′;



 



(SEQ ID NO: 139)



5′-TCAGCAGCAGTGGTGGT-3′;



 



(SEQ ID NO: 140)



5′-TAGAGCGCGGGGTTTCA-3′;



 



(SEQ ID NO: 141)



5′-TGAAAGCTCTGAAGAACTCAGTGAAAGCAGCTTT-3′;



 



(SEQ ID NO: 142)



5′-AATGCAGATGCCCTCCAC-3′;



 



(SEQ ID NO: 143)



5′-TCAGCAGCAGTGGTGGTGAAAGCTCTGAA-3′;



 



(SEQ ID NO: 144)



5′-TGTTCCAGGCGCCTG-3′;



 



(SEQ ID NO: 145)



5′-CACAGCTACAGATGCAAA-3′;



 



(SEQ ID NO: 146)



5′-GGTGCACACGGCTTTT-3′;



 



(SEQ ID NO: 147)



5′-TGTCCACAATTCTTCAGGCTTTTCATATCC-3′;



 



(SEQ ID NO: 148)



5′-TGGATATGAAAAGCCTGAAGTATTGTGGAC-3′;



 



(SEQ ID NO: 149)



5′-GGTCATTTACCATATGTACT-3′;



 



(SEQ ID NO: 150)



5′-AGCTACAGATGCAAANCAACACCACAGACA-3′;



 



(SEQ ID NO: 151)



5′-GAAAACTTTCCATTTAATGATGT-3′;



 



(SEQ ID NO: 152)



5′-ATTTTTTGATCTACCCTGGT-3′;



 



(SEQ ID NO: 153)



5′-TTGGTGGTCTGGGATGAAGG-3′;



 



(SEQ ID NO: 154)



5′-GTTTTATGGGCCGCCAAGTA-3′;



 



(SEQ ID NO: 155)



5′-TTCATCCCAGACCACCAAGG-3′;



 



(SEQ ID NO: 156)



5′-ATGGCTATTGCTAAAACTGTTCCAGTGTA-3′;



 



(SEQ ID NO: 157)



5′-TGGAATAATGAAAACTTTCCATTTAATGATGTAG-3′;



and



 



(SEQ ID NO: 158)



5′-CAATGGCCATTGCTAAAAGTGTTCCA-3′.






In some aspects, the present invention provides an isolated nucleic acid molecule that anneals under a stringent condition to a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 1, SEQ ID NO:128, SEQ ID NO:129, or a complement thereof, but does not anneal under the stringent condition to a non-parvovirus DNA or RNA molecule that may be present in a test sample (i.e., binds specifically to parvovirus B19 nucleic acid).
In one aspect, the present invention provides an isolated nucleic acid molecule that anneals under a stringent condition to a nucleotide sequence as set forth in SEQ ID NO: 1, SEQ ID NO:128, SEQ ID NO:129, or a complement thereof, wherein the isolated nucleic acid molecule also anneals under the stringent condition to a DNA or a RNA of parvovirus B19 Au, A6, V9, or D91.1.
In another aspect, the present invention provides an isolated nucleic acid molecule that anneals under a stringent condition to a nucleotide sequence or a complement thereof, wherein the nucleotide sequence is as set forth in SEQ ID NO: 1, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, and SEQ ID NO:133.
In other aspects, the present invention provides an isolated nucleic acid molecule that anneals under a stringent condition to a nucleotide sequence as set forth in SEQ ID NO: 1, SEQ ID NO:128, SEQ ID NO:129, or a complement thereof, wherein the isolated nucleic acid molecule does not anneal under the stringent condition to a DNA or a RNA of parvovirus B19 Au, A6, V9, or D91.1.
In various other aspects, the present invention provides an isolated nucleic acid molecule comprising an open reading frame, a partial open reading frame, or a complement thereof, wherein the open reading frame is comprised in SEQ ID NO: 1, SEQ ID NO:128, SEQ ID NO:129, or a complement thereof.
In other aspects, the present invention provides an isolated human erythrovirus comprising a genome comprising a nucleotide sequence as set forth in SEQ ID NO: 1, SEQ ID NO:128, or SEQ ID NO:129.
In one aspect, the present invention provides a kit comprising at least one primer and at least one probe, wherein the at least one primer comprises a primer nucleic acid sequence as set forth in (SEQ ID NO: 136), (SEQ ID NO: 137), (SEQ ID NO: 139), (SEQ ID NO: 140), (SEQ ID NO: 142), (SEQ ID NO: 144), (SEQ ID NO: 145), (SEQ ID NO: 146), (SEQ ID NO: 149), (SEQ ID NO: 151), (SEQ ID NO: 152), (SEQ ID NO: 154), or (SEQ ID NO: 155), wherein the at least one probe comprises a probe nucleic acid sequence as set forth in (SEQ ID NO: 138), (SEQ ID NO: 141), (SEQ ID NO: 143), (SEQ ID NO: 147), (SEQ ID NO: 148), (SEQ ID NO: 150), (SEQ ID NO: 153), (SEQ ID NO: 156), (SEQ ID NO: 157), (SEQ ID NO: 158), or complements thereof.
In another aspect, the kit comprises a forward primer, a reverse primer, and a probe, wherein the forward primer comprises a forward primer nucleic acid sequence as set forth in (SEQ ID NO: 136), (SEQ ID NO: 139), (SEQ ID NO: 142), (SEQ ID NO: 145), (SEQ ID NO: 149), (SEQ ID NO: 151), or (SEQ ID NO: 154), wherein the reverse primer comprises a reverse primer nucleic acid sequence as set forth in (SEQ ID NO: 137), (SEQ ID NO: 140), (SEQ ID NO: 144), (SEQ ID NO: 146), (SEQ ID NO: 152), or (SEQ ID NO: 155), wherein the probe comprises a probe nucleic acid sequence as set forth in (SEQ ID NO: 136), (SEQ ID NO: 137), (SEQ ID NO: 138), (SEQ ID NO: 139), (SEQ ID NO: 140), (SEQ ID NO: 141), (SEQ ID NO: 142), (SEQ ID NO: 143), (SEQ ID NO: 144), (SEQ ID NO: 145), (SEQ ID NO: 146), (SEQ ID NO: 147), (SEQ ID NO: 148), (SEQ ID NO: 149), (SEQ ID NO: 150), (SEQ ID NO: 151), (SEQ ID NO: 152), (SEQ ID NO: 153), (SEQ ID NO: 154), (SEQ ID NO: 155), (SEQ ID NO: 156), (SEQ ID NO: 157), (SEQ ID NO: 158), or complements thereof.
In some aspects, the present invention provides a method for detecting a parvovirus B19 in a sample. The method comprises:
a) performing PCR with at least a portion of the sample using a forward primer having a forward primer nucleic acid sequence and a reverse primer having a reverse primer nucleic acid sequence, wherein the forward primer nucleic acid sequence is as set forth in (SEQ ID NO: 136), (SEQ ID NO: 139), (SEQ ID NO: 142), (SEQ ID NO: 145), (SEQ ID NO: 149), (SEQ ID NO: 151), or (SEQ ID NO: 154), wherein the reverse primer nucleic acid sequence is as set forth in (SEQ ID NO: 137), (SEQ ID NO: 140), (SEQ ID NO: 144), (SEQ ID NO: 146), (SEQ ID NO: 152), or (SEQ ID NO: 155); and
b) determining the presence or absence of an amplicon, wherein the presence of the amplicon is indicative of the presence of the parvovirus B19 in the sample, wherein determining comprises annealing an oligonucleotide to the amplicon.
In further aspects, the present invention provides a method for determining parvovirus B19 in a sample. The method comprises
a) amplifying parvovirus B19 nucleic acid in the sample using at least one nucleic acid molecule comprising

    
    
        i) a sequence consisting of at least 12, at least 15, or at least 20 contiguous nucleotides or a complement thereof, wherein the contiguous nucleotides are contained in a nucleotide sequence selected from the group consisting of: (SEQ ID NO: 2), (SEQ ID NO: 3), (SEQ ID NO: 4), (SEQ ID NO: 5), (SEQ ID NO: 6), (SEQ ID NO: 7), (SEQ ID NO: 8), (SEQ ID NO: 9), (SEQ ID NO: 10), (SEQ ID NO: 11), (SEQ ID NO: 12), (SEQ ID NO: 13), (SEQ ID NO: 14), (SEQ ID NO: 15), (SEQ ID NO: 16), (SEQ ID NO: 17), (SEQ ID NO: 18), (SEQ ID NO: 19), (SEQ ID NO: 20), (SEQ ID NO: 21), (SEQ ID NO: 22), (SEQ ID NO: 23), (SEQ ID NO: 24), (SEQ ID NO: 25), (SEQ ID NO: 26), (SEQ ID NO: 27), (SEQ ID NO: 28), (SEQ ID NO: 29), (SEQ ID NO: 30), (SEQ ID NO: 31), (SEQ ID NO: 32), (SEQ ID NO: 33), (SEQ ID NO: 34), (SEQ ID NO: 35), (SEQ ID NO: 36), (SEQ ID NO: 37), (SEQ ID NO: 38), (SEQ ID NO: 39), (SEQ ID NO: 40), (SEQ ID NO: 41), (SEQ ID NO: 42), (SEQ ID NO: 43), (SEQ ID NO: 44), (SEQ ID NO: 45), (SEQ ID NO: 46), (SEQ ID NO: 47), (SEQ ID NO: 48), (SEQ ID NO: 49), (SEQ ID NO: 50), (SEQ ID NO: 51), (SEQ ID NO: 52), (SEQ ID NO: 53), (SEQ ID NO: 54), (SEQ ID NO: 55), (SEQ ID NO: 56), (SEQ ID NO: 57), (SEQ ID NO: 58), (SEQ ID NO: 59), and (SEQ ID NO: 60); or
        ii) at least 12, at least 15, or at least 20 contiguous nucleotides of a nucleotide sequence or a complement thereof, wherein the nucleotide sequence is selected from the group consisting of: (SEQ ID NO: 61), (SEQ ID NO: 62), (SEQ ID NO: 63), (SEQ ID NO: 64), (SEQ ID NO: 65), (SEQ ID NO: 66), (SEQ ID NO: 67), (SEQ ID NO: 68), (SEQ ID NO: 69), (SEQ ID NO: 70), (SEQ ID NO: 71), (SEQ ID NO: 72), (SEQ ID NO: 73), (SEQ ID NO: 74), (SEQ ID NO: 75), (SEQ ID NO: 76), (SEQ ID NO: 77), (SEQ ID NO: 78), (SEQ ID NO: 79), (SEQ ID NO: 80), (SEQ ID NO: 81), (SEQ ID NO: 82), (SEQ ID NO: 83), (SEQ ID NO: 84), (SEQ ID NO: 85), (SEQ ID NO: 86), (SEQ ID NO: 87), (SEQ ID NO: 88), (SEQ ID NO: 89), (SEQ ID NO: 90), (SEQ ID NO: 91), (SEQ ID NO: 92), (SEQ ID NO: 93), (SEQ ID NO: 94), (SEQ ID NO: 95), (SEQ ID NO: 96), (SEQ ID NO: 97), (SEQ ID NO: 98), (SEQ ID NO: 99), (SEQ ID NO: 100), (SEQ ID NO: 101), (SEQ ID NO: 102), (SEQ ID NO: 103), (SEQ ID NO: 104), (SEQ ID NO: 105), (SEQ ID NO: 106), (SEQ ID NO: 107), (SEQ ID NO: 108), (SEQ ID NO: 109), (SEQ ID NO: 110), (SEQ ID NO: 111), (SEQ ID NO: 112), (SEQ ID NO: 113), (SEQ ID NO: 114), (SEQ ID NO: 115), (SEQ ID NO: 116), (SEQ ID NO: 117), (SEQ ID NO: 118), (SEQ ID NO: 119), (SEQ ID NO: 120), (SEQ ID NO: 121), (SEQ ID NO: 122), (SEQ ID NO: 123), (SEQ ID NO: 124), (SEQ ID NO: 125), (SEQ ID NO: 126), and (SEQ ID NO: 127); and
    
    


b) detecting an amplicon generated in step (a), wherein detection of the amplicon indicates presence of the variant in the sample, wherein detecting optionally comprises the step of annealing at least one nucleic acid molecule of step (a)(ii) to the amplicon.
In some aspects, the present invention provides for a method for determining parvovirus B19 in a sample. The method comprises
a) amplifying parvovirus B19 nucleic acid in the sample using at least one nucleic acid molecule comprising a sequence consisting of at least 12, at least 15, or at least 20 contiguous nucleotides or a complement thereof, wherein the contiguous nucleotides are contained in a nucleotide sequence selected from the group consisting of: (SEQ ID NO: 2), (SEQ ID NO: 3), (SEQ ID NO: 4), (SEQ ID NO: 5), (SEQ ID NO: 6), (SEQ ID NO: 7), (SEQ ID NO: 8), (SEQ ID NO: 9), (SEQ ID NO: 10), (SEQ ID NO: 11), (SEQ ID NO: 12), (SEQ ID NO: 13), (SEQ ID NO: 14), (SEQ ID NO: 15), (SEQ ID NO: 16), (SEQ ID NO: 17), (SEQ ID NO: 18), (SEQ ID NO: 19), (SEQ ID NO: 20), (SEQ ID NO: 21), (SEQ ID NO: 22), (SEQ ID NO: 23), (SEQ ID NO: 24), (SEQ ID NO: 25), (SEQ ID NO: 26), (SEQ ID NO: 27), (SEQ ID NO: 28), (SEQ ID NO: 29), (SEQ ID NO: 30), (SEQ ID NO: 31), (SEQ ID NO: 32), (SEQ ID NO: 33), (SEQ ID NO: 34), (SEQ ID NO: 35), (SEQ ID NO: 36), (SEQ ID NO: 37), (SEQ ID NO: 38), (SEQ ID NO: 39), (SEQ ID NO: 40), (SEQ ID NO: 41), (SEQ ID NO: 42), (SEQ ID NO: 43), (SEQ ID NO: 44), (SEQ ID NO: 45), (SEQ ID NO: 46), (SEQ ID NO: 47), (SEQ ID NO: 48), (SEQ ID NO: 49), (SEQ ID NO: 50), (SEQ ID NO: 51), (SEQ ID NO: 52), (SEQ ID NO: 53), (SEQ ID NO: 54), (SEQ ID NO: 55), (SEQ ID NO: 56), (SEQ ID NO: 57), (SEQ ID NO: 58), (SEQ ID NO: 59), and (SEQ ID NO: 60); and
b) detecting an amplicon generated in step (a), wherein detection of the amplicon indicates presence of the parvovirus B19 in the sample, wherein detecting optionally comprises the step of annealing at least one nucleic acid molecule to the amplicon.
Advantages and benefits of the present invention will be apparent to one skilled in the art from reading this specification.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a partial genomic DNA sequence corresponding to a novel parvovirus B19 variant disclosed herein (i.e., SEQ ID NO: 1 for the D11 variant). Open reading frames (ORF) for VP1 and VP2 proteins begin at nucleotide position 2105 and 2786, respectively, whereas the partial ORF for NS 1 protein is shown from nucleotide position 1 through 2109, where nucleotide position 2107-2109, namely GAG, encodes glutamic acid.
FIG. 2A-2B shows a partial genomic DNA sequence corresponding to a novel parvovirus B19 variant disclosed and referred to herein as E3 (i.e., SEQ ID NO: 128). The open reading frame (ORF) for VP1 protein begins at nucleotide position 2141, whereas nucleotide position 1 through 2145 comprises the partial ORF for NS 1 protein.
FIG. 3A-3B shows a partial genomic DNA sequence corresponding to a novel parvovirus B19 variant disclosed and referred to herein as P1 (i.e., SEQ ID NO: 129). The open reading frame (ORF) for VP1 protein begins at nucleotide position 2263, whereas nucleotide position 1 through 2267 comprises the partial ORF for NS 1 protein.
FIG. 4A-U shows an alignment of partial DNA sequences of the novel human parvovirus B19 variants of the invention (i.e., SEQ ID NO: 1 for the D11 variant, SEQ ID NO: 128 for the E3 variant, SEQ ID NO: 129 for the P1 variant); Au human parvovirus B19 DNA, genotype 1 (i.e., SEQ ID NO: 130 for Accession Number: M13178); A6 human parvovirus B19 DNA, genotype 2 (i.e., SEQ ID NO: 131 for Accession Number: AY064476); V9 human parvovirus B19 DNA, genotype 3 (i.e., SEQ ID NO: 132 for Accession Number: NC—004295); and D91.1 human parvovirus B19 DNA, genotype 3 (i.e., SEQ ID NO: 133 for Accession Number: AY083234). In the alignment, the nucleotide shown at position 1 for SEQ ID NO:1, SEQ ID NO:128, and SEQ ID NO: 129 corresponds to the nucleotide at position 154 in FIG. 1, 991 in FIG. 2, and 1122 in FIG. 3, respectively.
FIG. 5A-E shows alignment of 97% consensus sequence to parvovirus B19 Genotype 1 prototype strain Au (i.e., PVBAUA, SEQ ID NO:130).
FIG. 6 shows alignment of the parvovirus B19 Genotype 1 prototype strain Au (i.e., PVBAUA, SEQ ID NO:130) with the 97% consensus sequence showing the highly conserved region from nucleotides 1351 to 2426.
FIG. 7 shows alignment of the parvovirus B19 Genotype 1 prototype strain Au (i.e., PVBAUA SEQ ID NO:130) with the 97% consensus sequence showing the highly conserved region from nucleotides 3704 to 4804.
FIG. 8 shows a graphical depiction of the parvovirus B19 genome and transcription map showing conserved sequences and target regions. ITR—Inverted Terminal Repeat, NS 1 Non-Structural Protein 1, VP1—Viral Protein 1, and VP2—Viral Protein 2.
FIG. 9 shows alignment of the primers and probes (SEQ ID NOs:154-158) of target region 5 with the parvovirus B19 genotype 1 Au isolate (nucleotides 1406-1559 of SEQ ID NO:130 of FIG. 4E are shown), the genotype 2 A6 isolate (nucleotides 1205-1358 of SEQ ID NO:131 of FIG. 4E are shown), and the genotype 3 isolates V9 (nucleotides 1298-1451 of SEQ ID NO:132 of FIG. 4E are shown) and D91.1 (nucleotides 1293-1446 of SEQ ID NO:133 of FIG. 4E are shown).
FIG. 10 shows target region and oligonucleotide sequences derived from highly conserved regions of the parvovirus B19 genome.
FIG. 11 shows average CT values for P1 (i.e., SEQ ID NO: 129) and E3 (i.e., SEQ ID NO: 128) across target regions 1 through 4. Test sample dilutions that showed no amplification and detection are plotted as CT=40.
FIG. 12 shows amplification plots for Primer/probe Set 2d at 60° C.: P1 (i.e., SEQ ID NO: 129) and E3 (i.e., SEQ ID NO: 128).
FIG. 13 shows amplification plots for Primer/probe Set 3b at 55° C.: P1 (i.e., SEQ ID NO: 129) and E3 (i.e., SEQ ID NO: 128).



DETAILED DESCRIPTION
It has been found in accordance with this invention novel human erythrovirus variants (herein named the “D11,” “P1,” or “E3” variant) that contain previously unreported variations in viral DNA sequence. While the new variants described herein share some nucleic acid homology with other previously known variants of the human erythrovirus family, regions of the viral genome show divergence. This divergence indicates three previously unknown human erythrovirus variants that have eluded current detection methods, and knowledge of these variants provides for new methods for viral screening and detection.
The term “human erythrovirus” herein refers to viral members of the genus Erythrovirus. 
The term “parvovirus B19” or “B19” herein refers to parvovirus B19 of the family Parvoviridae including genotypes 1, 2, and 3. For example, “parvovirus B19” at least includes parvovirus B19 genotype 1 (e.g., GenBank Accession Number: M13178) and related variants such as, for example, A6 parvovirus B19 genotype 2 (e.g., GenBank Accession Number: AY064476), V9 human parvovirus B19 genotype 3 (e.g., GenBank Accession Number: NC—004295), D91.1 parvovirus B19 genotype 3 (e.g., GenBank Accession Number: AY083234), and the parvovirus B19 variants (i.e., D11, E3, and P1) of the present invention. Fryer et al., (Emerg. Infect. Diseases 2006 12:151-154) discloses a phylogenetic analysis of members of the Parvoviridae subfamily including members of the Erythrovirus genus.
The term “universal base” herein refers to a moiety that may be substituted for any base. The “universal base” need not contribute to hybridization, but should not significantly detract from hybridization. Exemplary universal bases include, without limitation, inosine, 5-nitroindole and 4-nitrobenzimidazole.
In one embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 128, SEQ ID NO: 129, or a complement thereof. In some embodiments, the isolated nucleic acid molecule comprises single-stranded or double-stranded nucleic acid. In other embodiments, the isolated nucleic acid molecule comprises ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or any combination thereof.
The term “nucleic acid” or “nucleic acid molecule” herein broadly refers to a polymer of any size comprising RNA, DNA, modified RNA or DNA, spliced messenger RNA, RNA or DNA mimetics, or combinations thereof (e.g., RNA/DNA hybrids). The term, therefore, includes polymers composed of naturally-occurring nucleotide bases, sugars and covalent internucleoside (backbone) linkages as well as nucleic acid molecules having non-naturally-occurring portions that function similarly. Further, the term “nucleic acid molecule” also includes polymers that are double-stranded, single-stranded, or any combination thereof.
In another aspect of the invention, an isolated nucleic acid molecule is provided that can anneal to DNA or RNA of the novel parvovirus B19 variant (i.e., D11) of the invention, as well as to one or more of other parvovirus sequences such as, for example, sequences of Au, A6, V9, or D91.1 parvovirus, thereby providing probes and primers useful for detection of multiple forms in a single test. Thus, according to this aspect, the invention relates to nucleic acid molecules useful for detection of parvovirus B19, including the novel variant characterized by the nucleic acid sequence of SEQ ID NO:1, and one or more variants known as Au, A6, V9, and D91.1 (see FIG. 4). Accordingly, the present invention includes an isolated nucleic acid molecule comprising a sequence consisting of at least 12, at least 15, or at least 20 contiguous nucleotides or a complement thereof, wherein the contiguous nucleotides are contained in a nucleotide sequence selected from the group consisting of: (SEQ ID NO: 2), (SEQ ID NO: 3), (SEQ ID NO: 4), (SEQ ID NO: 5), (SEQ ID NO: 6), (SEQ ID NO: 7), (SEQ ID NO: 8), (SEQ ID NO: 9), (SEQ ID NO: 10), (SEQ ID NO: 11), (SEQ ID NO: 12), (SEQ ID NO: 13), (SEQ ID NO: 14), (SEQ ID NO: 15), (SEQ ID NO: 16), (SEQ ID NO: 17), (SEQ ID NO: 18), (SEQ ID NO: 19), (SEQ ID NO: 20), (SEQ ID NO: 21), (SEQ ID NO: 22), (SEQ ID NO: 23), (SEQ ID NO: 24), (SEQ ID NO: 25), (SEQ ID NO: 26), (SEQ ID NO: 27), (SEQ ID NO: 28), (SEQ ID NO: 29), (SEQ ID NO: 30), (SEQ ID NO: 31), (SEQ ID NO: 32), (SEQ ID NO: 33), (SEQ ID NO: 34), (SEQ ID NO: 35), (SEQ ID NO: 36), (SEQ ID NO: 37), (SEQ ID NO: 38), (SEQ ID NO: 39), (SEQ ID NO: 40), (SEQ ID NO: 41), (SEQ ID NO: 42), (SEQ ID NO: 43), (SEQ ID NO: 44), (SEQ ID NO: 45), (SEQ ID NO: 46), (SEQ ID NO: 47), (SEQ ID NO: 48), (SEQ ID NO: 49), (SEQ ID NO: 50), (SEQ ID NO: 51), (SEQ ID NO: 52), (SEQ ID NO: 53), (SEQ ID NO: 54), (SEQ ID NO: 55), (SEQ ID NO: 56), (SEQ ID NO: 57), (SEQ ID NO: 58), (SEQ ID NO: 59), (SEQ ID NO: 60), (SEQ ID NO: 134), and (SEQ ID NO: 135).
In another aspect of the invention, an isolated nucleic acid molecule is provided that can anneal to DNA or RNA of the novel parvovirus B19 variants (i.e., D11, E3 and P1) of the invention, as well as to one or more other parvovirus sequences such as, for example, sequences of Au, A6, V9, or D91.1 parvovirus, thereby providing probes and primers useful for detection of multiple forms in a single test. Thus, according to this aspect, the invention relates to nucleic acid molecules useful for detection of parvovirus B19, including one or more of the novel variants characterized by the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:128, and SEQ ID NO: 129, and one or more variants known as Au, A6, V9, and D91.1. Accordingly, the present invention includes an isolated nucleic acid molecule comprising a sequence, or a complement thereof, present in an erythrovirus genome comprising a nucleotide sequence as set forth in SEQ ID NO:1, SEQ ID NO:128, and SEQ ID NO: 129, wherein the sequence is present in at least one other parvovirus genome. In one embodiment, the at least one other parvovirus genome is parvovirus B19 Au, A6, V9, and D91.1.
In another embodiment, the present invention includes an isolated nucleic acid molecule comprising a sequence consisting of at least 12, at least 15, or at least 20 contiguous nucleotides or a complement thereof, wherein the contiguous nucleotides are contained in a nucleotide sequence selected from the group consisting of: (SEQ ID NO: 13), (SEQ ID NO: 14), (SEQ ID NO: 15), (SEQ ID NO: 16), (SEQ ID NO: 17), (SEQ ID NO: 18), (SEQ ID NO: 19), (SEQ ID NO: 20), (SEQ ID NO: 21), (SEQ ID NO: 22), (SEQ ID NO: 23), (SEQ ID NO: 25), (SEQ ID NO: 26), (SEQ ID NO: 27), (SEQ ID NO: 28), (SEQ ID NO: 29), (SEQ ID NO: 30), (SEQ ID NO: 31), (SEQ ID NO: 32), (SEQ ID NO: 33), (SEQ ID NO: 34), (SEQ ID NO: 35), (SEQ ID NO: 36), (SEQ ID NO: 37), (SEQ ID NO: 38), (SEQ ID NO: 134), and (SEQ ID NO: 135).
In other embodiments, the present invention provides an isolated nucleic acid molecule consisting of a nucleotide sequence or a complement thereof, wherein the nucleotide sequence is selected from the group consisting of: (SEQ ID NO: 136), (SEQ ID NO: 137), (SEQ ID NO: 138), (SEQ ID NO: 139), (SEQ ID NO: 140), (SEQ ID NO: 141), (SEQ ID NO: 142), (SEQ ID NO: 143), (SEQ ID NO: 144), (SEQ ID NO: 145), (SEQ ID NO: 146), (SEQ ID NO: 147), (SEQ ID NO: 148), (SEQ ID NO: 149), (SEQ ID NO: 150), (SEQ ID NO: 151), (SEQ ID NO: 152), (SEQ ID NO: 153), (SEQ ID NO: 154), (SEQ ID NO: 155), (SEQ ID NO: 156), (SEQ ID NO: 157), and (SEQ ID NO: 158).
In other aspects, the isolated nucleic acid molecules allow for discrimination between the newly discovered D11 variant of the present invention and other known parvovirus B19 such as, for example, Au, A6, V9, and D91.1. Thus, the present invention provides an isolated nucleic acid molecule comprising at least 12, at least 15, or at least 20, contiguous nucleotides of a nucleotide sequence or its complement thereof. The nucleotide sequence is based on regions within the genome of the newly discovered D11 variant of the present invention. The at least 12, at least 15, or at least 20, contiguous nucleotides can provide for specific and/or sensitive detection of a human erythrovirus in a sample. Accordingly, the present invention includes an isolated nucleic acid molecule comprising at least 12, at least 15, or at least 20 contiguous nucleotides of a nucleotide sequence or a complement thereof, wherein the nucleotide sequence is selected from the group consisting of: (SEQ ID NO: 61), (SEQ ID NO: 62), (SEQ ID NO: 63), (SEQ ID NO: 64), (SEQ ID NO: 65), (SEQ ID NO: 66), (SEQ ID NO: 67), (SEQ ID NO: 68), (SEQ ID NO: 69), (SEQ ID NO: 70), (SEQ ID NO: 71), (SEQ ID NO: 72), (SEQ ID NO: 73), (SEQ ID NO: 74), (SEQ ID NO: 75), (SEQ ID NO: 76), (SEQ ID NO: 77), (SEQ ID NO: 78), (SEQ ID NO: 79), (SEQ ID NO: 80), (SEQ ID NO: 81), (SEQ ID NO: 82), (SEQ ID NO: 83), (SEQ ID NO: 84), (SEQ ID NO: 85), (SEQ ID NO: 86), (SEQ ID NO: 87), (SEQ ID NO: 88), (SEQ ID NO: 89), (SEQ ID NO: 90), (SEQ ID NO: 91), (SEQ ID NO: 92), (SEQ ID NO: 93), (SEQ ID NO: 94), (SEQ ID NO: 95), (SEQ ID NO: 96), (SEQ ID NO: 97), (SEQ ID NO: 98), (SEQ ID NO: 99), (SEQ ID NO: 100), (SEQ ID NO: 101), (SEQ ID NO: 102), (SEQ ID NO: 103), (SEQ ID NO: 104), (SEQ ID NO: 105), (SEQ ID NO: 106), (SEQ ID NO: 107), (SEQ ID NO: 108), (SEQ ID NO: 109), (SEQ ID NO: 110), (SEQ ID NO: 111), (SEQ ID NO: 112), (SEQ ID NO: 113), (SEQ ID NO: 114), (SEQ ID NO: 115), (SEQ ID NO: 116), (SEQ ID NO: 117), (SEQ ID NO: 118), (SEQ ID NO: 119), (SEQ ID NO: 120), (SEQ ID NO: 121), (SEQ ID NO: 122), (SEQ ID NO: 123), (SEQ ID NO: 124), (SEQ ID NO: 125), (SEQ ID NO: 126), and (SEQ ID NO: 127).
In one embodiment, the nucleotide sequence is selected from the group consisting of (SEQ ID NO: 61), (SEQ ID NO: 64), (SEQ ID NO: 65), (SEQ ID NO: 66), (SEQ ID NO: 68), (SEQ ID NO: 70), (SEQ ID NO: 81), (SEQ ID NO: 84), (SEQ ID NO: 86), (SEQ ID NO: 87), (SEQ ID NO: 88), (SEQ ID NO: 89), (SEQ ID NO: 97), (SEQ ID NO: 102), (SEQ ID NO: 109), SEQ ID NO: 114), (SEQ ID NO: 119), (SEQ ID NO: 120), (SEQ ID NO: 121), and (SEQ ID NO: 127).
In other aspects, the isolated nucleic acid molecules allow for discrimination between the newly discovered variants of the present invention (i.e., D11, E3, and P1) and at least one other known parvovirus B19 such as, for example, Au, A6, V9, and D91.1. Thus, the present invention provides an isolated nucleic acid molecule comprising a sequence present in an erythrovirus genome or a complement thereof, wherein the genome comprises a nucleotide sequence as set forth in SEQ ID NO:1, SEQ ID NO:128 or SEQ ID NO: 129, wherein the sequence is not present in at least one other parvovirus genome. The nucleotide sequence is based on regions of the genome of the newly discovered D11, E3 and/or P1 variants of the present invention. The contiguous nucleotides can provide for specific and/or sensitive detection of a human erythrovirus in a sample.
In other aspects, the present invention provides an isolated nucleic acid molecule that anneals under a stringent condition to a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 1 or a complement thereof, but does not anneal under the stringent condition to other DNA or RNA molecules that may be present in a test sample (i.e., binds specifically to parvovirus B19 nucleic acid). In one embodiment, the nucleotide sequence is as set forth in SEQ ID NO: 128 or a complement thereof. In another embodiment, the nucleotide sequence is as set forth in SEQ ID NO: 129 or a complement thereof. As used herein, a stringent condition is a highly stringent condition or a moderately stringent condition.
Stringency conditions are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology (1999. Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K, editors. John Wiley & Sons, Inc.) or Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press (1989). Stringency conditions relate to the set of conditions under which nucleic acid hybrids comprising double-stranded regions are formed and/or maintained. It is well known in the art that two complementary single-stranded nucleic acids (DNA or RNA) can anneal to one another so that complexes called hybrids are formed. Formation or subsequent stability of a formed hybrid can be affected by the conditions under which hybridization (i.e., annealing) occurs, by any wash conditions subsequent to hybridization, or both. Thus, through one or more nucleic acid hybridization steps, which can precede one or more wash steps, two nucleic acid sequences having a certain degree of complementary identity to one another can anneal together and form a hybrid comprising one or more contiguous regions of double-stranded nucleic acid. Further, formation of hybrids can occur in a variety of environments such as, for example, in solution, with one component immobilized on a solid support such as a nylon membrane, nitrocellulose paper, polystyrene, or in situ (e.g., in suitably prepared cells or histological sections).
It is well known in the art that a number of factors affect hybrid formation and/or stability such as, for example, temperature, duration, frequency, or salt or detergent concentration of the hybridization and/or wash conditions. Thus, for example, the stringency of a condition can be primarily due to the wash conditions, particularly if the hybridization condition used is one which allows less stable hybrids to form along with stable hybrids (e.g., wash conditions at higher stringency can remove less stable hybrids). In general, longer sequences require higher temperatures for proper annealing, while shorter sequences need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acids to reanneal when complementary strands are present in a favorable environment at temperatures below their melting temperature. The higher the degree of desired homology between two sequences, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so.
Generally, stringency can be altered or controlled by, for example, manipulating temperature and salt concentration during hybridization and washing. For example, a combination of high temperature and low salt concentration increases stringency. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. of the stringent condition as necessary to accommodate factors such as polynucleotide length and the like.
A “highly stringent condition,” as defined herein, can be identified by a condition that comprises 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a wash consisting of 0.1×SSC at 55° C.
A “moderately stringent condition,” as defined herein, can be identified by washing and/or hybridization conditions less stringent than those described above for a highly stringent condition. An example of a moderately stringent condition is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing in 1×SSC at about 35-50° C.
Sometimes, nucleic acid duplex or hybrid stability is expressed as the melting temperature or Tm, which is the temperature at which 50% of one nucleic acid dissociates from a nucleic acid duplex. Accordingly, this melting temperature can be used to define the required stringency conditions. If sequences are related and substantially identical to each other, rather than identical, then it can be useful to first establish the lowest temperature at which only homologous annealing occurs with a particular concentration of salt (e.g., SSC or SSPE). Then, assuming 1% mismatching results in a 1° C. decrease in the Tm, the temperature of the final wash in the hybridization reaction is reduced accordingly (for example, if sequences having >95% identity with each other are sought, the final wash temperature is decreased by 5° C.). In practice, the change in Tm can be between 0.5° C. and 1.5° C. per 1% mismatch.
In other aspects, the present invention provides an isolated nucleic acid molecule that anneals under a highly stringent condition to a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 1 or a complement thereof, wherein the isolated nucleic acid molecule does not anneal under the highly stringent condition to a DNA or a RNA of parvovirus B19 Au, A6, V9, or D91.1 variant. In one embodiment, the polynucleotide comprises the nucleotide sequence as set forth in SEQ ID NO: 128 or a complement thereof. In another embodiment, the polynucleotide comprises the nucleotide sequence as set forth in SEQ ID NO: 129 or a complement thereof. Stringency conditions are as described above.
The annealing portion of a hybridizing nucleic acid molecule can vary in length but is typically at least about 6, illustratively, at least about 10, 12, 15, 20, 25, 30, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length. However, binding enhancers such as minor groove binding nucleic acids can allow annealing to shorter nucleic acid targets with increased sequence-specificity compared to ordinary length nucleic acids (Kutyavin I V, et al., Nucleic Acid Research 2000 28:655-661). The annealing portion of the annealing nucleic acid is at least 60%, e.g., at least 70%, 80%, 95% or at least 98% identical to the sequence of a portion or all of a nucleic acid expressly described herein, or its complement. Annealing nucleic acids of the type described herein can be used, for example, as cloning probes, primers (e.g., a PCR primer), or diagnostic probes.
As described above, oligonucleotide primers and probes can be derived from the nucleic acid sequences disclosed herein. In various embodiments, primers and probes are used in combination with each other. The present invention finds use in a variety of different applications including, but not limited to, research, medical, and diagnostic applications.
In some embodiments, primers and probes can be designed from regions of SEQ ID NO: 1, SEQ ID NO: 128, or SEQ ID NO:129, wherein the primers and probes each comprise one or more conserved nucleotides also present in a corresponding region of the genome of another human erythroviruses such as parvovirus B19 Au, A6, V9, and D91.1. For example, a nucleotide sequence alignment can be performed with at least two parvovirus sequences using, e.g., a computer algorithm to determine identical contiguous nucleotide sequences common to the at least two parvovirus. Accordingly, the primers and probes can provide for reagents for use in, for example, a parvovirus detection assay or kit thereby expanding the repertoire of parvovirus variants that can be detected by the assay or kit.
In another embodiment, the present invention provides a kit comprising at least one primer and at least one probe, wherein the at least one primer comprises a primer nucleic acid sequence as set forth in (SEQ ID NO: 136), (SEQ ID NO: 137), (SEQ ID NO: 139), (SEQ ID NO: 140), (SEQ ID NO: 142), (SEQ ID NO: 144), (SEQ ID NO: 145), (SEQ ID NO: 146), (SEQ ID NO: 149), (SEQ ID NO: 151), (SEQ ID NO: 152), (SEQ ID NO: 154), or (SEQ ID NO: 155), wherein the at least one probe comprises a probe sequence consisting of: (SEQ ID NO: 138), (SEQ ID NO: 141), (SEQ ID NO: 143), (SEQ ID NO: 147), (SEQ ID NO: 148), (SEQ ID NO: 150), (SEQ ID NO: 153), (SEQ ID NO: 156), (SEQ ID NO: 157), (SEQ ID NO: 158), or complements thereof.
In another embodiment, the kit comprises a forward primer, a reverse primer, and a probe, wherein the forward primer comprises a forward primer nucleic acid sequence as set forth in (SEQ ID NO: 136), (SEQ ID NO: 139), (SEQ ID NO: 142), (SEQ ID NO: 145), (SEQ ID NO: 149), (SEQ ID NO: 151), or (SEQ ID NO:154, wherein the reverse primer comprises a reverse primer nucleic acid sequence as set forth in (SEQ ID NO: 137), (SEQ ID NO: 140), (SEQ ID NO: 144), (SEQ ID NO: 146), (SEQ ID NO: 152), or (SEQ ID NO:155, wherein the probe comprises a probe nucleic acid sequence as set forth in (SEQ ID NO: 136), (SEQ ID NO: 137), (SEQ ID NO: 138), (SEQ ID NO: 139), (SEQ ID NO: 140), (SEQ ID NO: 141), (SEQ ID NO: 142), (SEQ ID NO: 143), (SEQ ID NO: 144), (SEQ ID NO: 145), (SEQ ID NO: 146), (SEQ ID NO: 147), (SEQ ID NO: 148), (SEQ ID NO: 149), (SEQ ID NO: 150), (SEQ ID NO: 151), (SEQ ID NO: 152), (SEQ ID NO: 153), (SEQ ID NO: 154), (SEQ ID NO: 155), (SEQ ID NO: 156), (SEQ ID NO: 157), (SEQ ID NO: 158), or complements thereof. In some embodiments, the probe nucleic acid sequence is as set forth in (SEQ ID NO: 138), (SEQ ID NO: 141), (SEQ ID NO: 143), (SEQ ID NO: 147), (SEQ ID NO: 148), (SEQ ID NO: 150), (SEQ ID NO: 153), (SEQ ID NO: 156), (SEQ ID NO: 157), (SEQ ID NO: 158), or complements thereof.
In some aspects, the present invention provides a method for detecting a parvovirus B19 in a sample. The method comprises:
a) performing PCR with at least a portion of the sample using a forward primer having a forward primer nucleic acid sequence and a reverse primer having a reverse primer nucleic acid sequence, wherein the forward primer nucleic acid sequence is as set forth in (SEQ ID NO: 136), (SEQ ID NO: 139), (SEQ ID NO: 142), (SEQ ID NO: 145), (SEQ ID NO: 149), (SEQ ID NO: 151), or (SEQ ID NO:154, wherein the reverse primer nucleic acid sequence is as set forth in (SEQ ID NO: 137), (SEQ ID NO: 140), (SEQ ID NO: 144), (SEQ ID NO: 146), (SEQ ID NO: 152), or (SEQ ID NO:155); and
b) determining the presence or absence of an amplicon, wherein the presence of the amplicon is indicative of the presence of the parvovirus B19 in the sample. In one embodiment, determining comprises annealing an oligonucleotide to the amplicon, wherein the oligonucleotide comprises a sequence as set forth in (SEQ ID NO: 138), (SEQ ID NO: 141), (SEQ ID NO: 143), (SEQ ID NO: 147), (SEQ ID NO: 148), (SEQ ID NO: 150), (SEQ ID NO: 153); (SEQ ID NO: 156), (SEQ ID NO: 157), (SEQ ID NO: 158), or complements thereof. In one embodiment, the oligonucleotide further comprises a detectable label. In another embodiment, the PCR is a real-time PCR.
In some embodiments, primers and probes can be designed from regions of SEQ ID NO: 1, SEQ ID NO: 128, or SEQ ID NO:129 that comprise one or more unique nucleotides as compared to the corresponding region of the genome of other human erythroviruses such as parvovirus B19 Au, A6, V9, and D91.1. Accordingly, the primers and probes can provide for assays that facilitate detection of a parvovirus B19 as well as provide for assays that distinguish between one or more of the new variants disclosed herein or other previously known human erythroviruses such as parvovirus B19 Au, A6, V9, and D91.1. Accordingly, the primers and probes can provide for a more specific parvovirus detection assay that can discriminate from among the parvovirus variants.
Another example of a hybridization assay probe is a structure referred to as a “molecular beacon,” which is described, for example, in U.S. Pat. No. 5,925,517. Molecular beacons are oligonucleotide hybridization probes that comprise a label pair and form a stem-and-loop structure. The loop component comprises a probe sequence that is complementary to a target sequence. The stem comprises an affinity pair (or nucleic acid arms) that hold the probe in a closed conformation in the absence of a target nucleic acid sequence. The stem is formed by the annealing of complementary arm sequences that are located on either side of the probe sequence. Hybridization of the target nucleic acid and the target complement sequence separates the members of the affinity pair, thereby shifting the probe to an open conformation. The shift to the open confirmation is detectable due to reduced interaction of the label pair, which may be, for example, a fluorophore and a quencher (e.g., DABCYL and EDANS).
The simultaneous use of two or more probes using donor-acceptor energy transfer is known in the art. Accordingly, molecular beacons can be synthesized that possess differently colored fluorophores, enabling assays to be carried out that simultaneously detect different targets in the same reaction. For example, multiplex assays can contain a number of different primer sets, each set enabling the amplification of a unique gene sequence from a different pathogenic agent, and a corresponding number of molecular beacons can be present, each containing a probe sequence specific for one of the amplicons, and each labeled with a fluorophore of a different color. The color of the resulting fluorescence, if any, identifies the pathogenic agent in the sample, and the number of amplification cycles required to generate detectable fluorescence provides a quantitative measure of the number of target organisms present. If more than one type of pathogen is present in the sample, the fluorescent colors that occur identify which are present. Moreover, due to the inherent design of gene amplification assays, the use of molecular beacons enables the abundance of a rare pathogen to be determined in the presence of a much more abundant pathogen.
In general, primers can provide for specific amplification (e.g., by PCR) of a target nucleic acid to produce an amplification product (also referred to as an “amplicon”). In one embodiment, the target nucleic acid is DNA or RNA of the novel human erythrovirus disclosed herein. In some embodiments, the target nucleic acid comprises a genome comprising a nucleotide sequence as set forth in SEQ ID NO: 1. In other embodiments, the target nucleic acid comprises an RNA molecule transcribed from a genome comprising a nucleotide sequence as set forth in SEQ ID NO: 1. In one embodiment, the genome comprises the nucleotide sequence as set forth in SEQ ID NO: 128 or SEQ ID NO: 129.
In some embodiments, a primer sequence can be at least about 10 nucleotides in length, illustratively about 10 to about 100, about 12 to about 75, about 14 to about 65, about 16 to about 60, about 20 to about 55, about 25 to about 50, or about 30 to about 45, and the like. In one embodiment, a primer sequence is about 15 to about 20 nucleotides in length.
Probes are generally designed so as to have a nucleotide sequence complementary to one or more variant nucleotides within a target region sequence. Probes suitable for use in amplification-based detection methods can be designed from any sequence positioned within the sequence of an amplification product that would be produced using two selected primers. In various embodiments, a probe sequence can be at least about 10 nucleotides in length, illustratively about 10 to about 100, about 12 to about 75, about 14 to about 65, about 16 to about 60, about 20 to about 55, about 25 to about 50, or about 30 to about 45, and the like. In one embodiment, a probe sequence is about 15 to about 20 nucleotides in length.
Depending on the number of nucleotide residues, a nucleic acid molecule also can be referred to as an “oligonucleotide” or “oligomer.” The term “oligonucleotide” or “oligomer” typically refers to a nucleic acid molecule having a relatively short sequence of nucleotides. For example, an oligonucleotide can be about 5 to about 500 nucleotide residues in length. Oligonucleotides, which can be double-stranded or single-stranded, may be used as single-stranded probes for detecting complementary DNA or RNA because they can bind readily to their complements. Non-limiting examples of procedures that use oligonucleotides are nucleic acid testing (NAT), DNA microarrays, amplified fragment-length polymorphism (AFLP) analysis, fragment analysis, Southern blots, and fluorescent in situ hybridization (FISH). Oligonucleotides composed of DNA are often used in the polymerase chain reaction (PCR), a procedure well-known to those skilled in the art. In this regard, the oligonucleotide also can be referred to as a “primer,” which is a short piece of DNA that binds to its complementary target sequence. This generates a place for a polymerase to bind and extend the primer by the addition of nucleotides to make a complementary copy of the target sequence. An oligonucleotide can also be referred to as a “probe” which is a short piece of DNA or RNA that can be used to detect and identify specific DNA or RNA molecules bearing the complementary sequence. Probe detection is achieved through fluorescence, colorimetry, radioactivity, antigen binding, or enzymatic activity.
In some embodiments, the isolated nucleic acid molecule is an oligonucleotide having at least about 5 nucleotide residues in length, illustratively about 5 to about 500, about 8 to about 400, about 10 to about 300, about 12 to about 200, about 14 to about 100, about 16 to about 90, about 18 to about 80, about 20 to about 70, about 25 to about 60, or about 30 to about 50.
One skilled in the art will appreciate that the isolated nucleic acid molecules of the present invention can be obtained by standard molecular biology techniques such as PCR and others described in Current Protocols in Molecular Biology (1999. Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K, editors. John Wiley & Sons, Inc.) or by chemical synthesis or by nucleic acid analogs.
Methods involving chemical synthesis may be automated and commercially available and can include, for example, phosphodiester, phosphotriester, or phosphoramidite methods. U.S. Pat. Nos. 4,458,066; 4,415,732; and Meth. Enzymol. 1979 68:90 and 109, which are incorporated herein by reference, disclose examples of chemical synthesis methods. Chemical nucleic acid synthesis allows for the incorporation of unnatural or modified bases, as well as a variety of labeling moieties, into a nucleic acid molecule. Further, modified backbone chemistries such as, for example, peptide linkages, phosphorothioates, phosphoroamidates, phosphotriesters, 2′-O-Methyl RNA, 2′-O-Mt RNA, P-Ethoxy DNA, and P-Ethoxy 2′-O-Mt RNA are also readily available and known in the art. Furthermore, the uses of cross-linkable probes in nucleic acid hybridization assays to cross-link to target sequences are known in the art. For example, compounds based on furocoumarin or psoralen attached to nucleic acid molecules through adduct formation are described in U.S. Pat. No. 4,826,967 and U.S. Pat. No. 5,082,934, both incorporated herein by reference, describes a photoactivatable nucleoside analogue comprising a coumarin moiety linked through its phenyl ring to the 1-position of a ribose or deoxyribose sugar moiety in the absence of an intervening base moiety.
Nucleic acid analogs and mimics have similar chemical structures as native nucleic acid molecules but with unique modifications. Nucleic acid analogs, such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos, improve the capabilities of traditional nucleic acid molecules beyond the limitations associated with standard nucleic acids chemistry (Karkare S and Bhatnagar D. Appl. Microbiol. Biotechnol. 2006 71:575-586.) Such nucleic acid analogs greatly expand and improve the capabilities to detect and identify related nucleic acid sequences.
In some aspects, an isolated nucleic acid molecule of the present invention further comprises one or more heterologous nucleotides. The term “heterologous nucleotides” herein refers to a nucleotide or nucleotides that are not a natural part of the isolated nucleic acid molecule but which are naturally or artificially joined to the isolated nucleic acid molecule. Examples of a heterologous nucleic acid sequence include, but is not limited to, a vector sequence, a sequence that is complementary to a base sequence of a purification probe, a control sequence such as, for example, an enhancer or a promoter sequence (i.e., a sequence that is recognized by an RNA polymerase that binds to that sequence and initiates transcription to produce RNA transcripts), and a sequence comprising one or more restriction enzyme sites.
The term “control sequence” herein refers to sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, messenger RNA splicing signals, polyadenylation signals, and enhancers.
A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
In one embodiment, the one or more heterologous nucleotides comprise a sequence that is complementary to a base sequence of a purification probe. The purification probe can be joined to solid supports such as, for example, a matrix or particles free in solution. Non-limiting examples of a solid support include nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane polypropylene, and magnetically-attractable particles. For example, the purification probe, which may comprise a DNA or RNA sequence, can be labeled with amine or biotin tags via a cross-linker. These biotin or amine labeled purification probes are then amenable to immobilization and detection strategies that allow in vitro nucleic acid:nucleic acid or protein:nucleic acid interactions. Thus, annealing of the heterologous segment of the isolated nucleic acid molecule with its complementary base sequence of the purification probe can facilitate sample purification of molecules that anneal to the virus-specific sequence segment of the isolated nucleic acid molecule. U.S. Pat. No. 6,534,273, incorporated herein by reference, describes a method for capturing a target nucleic acid molecule in a sample onto a solid support.
In one embodiment, the isolated nucleic acid molecules of the present invention are joined to a solid support such as those described above.
In some embodiments, the one or more heterologous nucleotides comprise one or more repeating base sequences, for example, one or more repeating base sequences that are complementary to one or more repeating base sequences of the purification probe. A repeating base sequences can be a regularly repeating base sequence, such as those formed, for example, by nucleic acid homopolymers of poly-adenine (An), poly-thymine (Tn), poly-cytosine (Cn), poly-guanine (Gn), and poly-uridine (Un). Repeating sequences also can include mixed polymers, such as AT repeats ([AT]n), and the like.
The number of bases of the repeating base sequence of the one or more heterologous nucleotides of the isolated nucleic acid molecule can be equal to, greater than, or less than the number of bases of the repeating base sequence of the purification probe. The lengths of the complementary repeating sequences can determine the melting temperature (Tm) of the heterologous segment:purification probe complex. In one embodiment, the repeating base sequence of the heterologous segment is longer than the complementary repeating base sequence of the purification probe. In another embodiment, the repeating base sequence of the heterologous segment or the purification probe can be at least about 5 bases in length, illustratively about 5 to about 40, about 10 to about 30, or about 15 to about 20, and the like.
In other embodiments, the one or more heterologous nucleotides comprise an operably linked control sequence. In one embodiment, the control sequence is an enhancer or a promoter sequence that is specifically recognized by an RNA polymerase that binds to that sequence and initiates transcription to produce RNA transcripts. Non-limiting examples of promoters recognized by an RNA polymerase include promoters such as T3, T7, or SP6. Thus, an isolated nucleic acid molecule can be used in a variety of nucleic acid based assays including assays that use an RNA polymerase to produce multiple RNA transcripts such as, for example, transcription-mediated amplification (TMA) assay as described in Nature 350:91-92 (1991); and U.S. Pat. No. 5,399,491, both incorporated herein by reference.
Optionally, the isolated nucleic acid molecules of the present invention can be coupled to a label that can be detected. The label can be joined directly or indirectly to the isolated nucleic acid molecule. The labeling of a nucleic acid can be performed by covalently attaching a detectable group (label) to either an internal or terminal position, for example. One skilled in the art knows that there are a variety of ways for derivatizing oligonucleotides with reactive functionalities that permit the addition of a label. A number of approaches are available for directly attaching labels to nucleic acid molecules and for biotinylating probes so that radioactive, fluorescent, chemiluminescent, enzymatic, or electron dense labels can be attached via avidin. Non-limiting examples of references describing labels and methods for labeling nucleic acids include U.S. Pat. No. 4,605,735; U.S. Pat. No. 4,757,141; U.S. Pat. No. 6,965,020; Nucl. Acids Res. 5:363 (1978); Nucl. Acids Res. 13:1529 (1985); Nucl. Acids Res. 15:3131 (1987); Nucl. Acids Res. 15:6455 (1987); Nucl. Acids Res. 13:4485 (1985); Nucl. Acids Res. 15:4837 (1987); and Anal. Biochem. 169:1-25 (1988), which are incorporated herein by reference for their disclosure relating to labeling of nucleic acids.
The isolated nucleic acid molecules of the present invention may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
Further, a viral protein encoded by an isolated nucleic acid molecule comprising an open reading frame or a complement thereof, wherein the open reading frame is comprised in SEQ ID NO: 1, SEQ ID NO 128, or SEQ ID NO: 129 may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the protein-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, a factor leader (including Saccharomyces and Kluyveromyces α-factor leaders, the latter described in U.S. Pat. No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179), or the signal described in WO/90/13646. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. 
An example of suitable selectable markers for mammalian cells is those that enable the identification of cells competent to take up the viral protein-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Proc. Natl. Acad. Sci. USA 77:4216 (1980). A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 as described by Nature 282:39 (1979); Gene 7:141 (1979); and Gene 10:157 (1980). The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 as described by Genetics 85:12 (1977).
Expression and cloning vectors usually contain a promoter operably linked to the viral-protein-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems as described by Nature 275:615 (1978) and Nature 281:544 (1979), alkaline phosphatase, a tryptophan (trp) promoter system as described by Nucl. Acids Res. 8:4057 (1980) and EP 36,776, and hybrid promoters such as the tac promoter as described in Proc. Natl. Acad. Sci. USA 80:21 25 (1983). Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the viral protein.
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase described in J. Biol. Chem. 255:2073 (1980) or other glycolytic enzymes described in J. Adv. Enzyme Reg. 7:149 (1968) and Biochemistry 17:4900 (1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
Viral protein transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (GB 2,211,504), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-B virus, and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
Transcription of a DNA encoding the viral protein by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to about 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the CMV early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5′ or 3′ to the viral protein coding sequence, but is preferably located at a site 5′ from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly upstream of the polyadenylation site in untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the viral protein.
Other methods, vectors, and host cells suitable for adaptation to the synthesis of viral proteins in recombinant vertebrate cell culture are described in Nature 293:620 625 (1981); Nature 281:4046 (1979); EP 117,060; and EP 117,058.
A host cell can be transfected or transformed with the isolated nucleic acid molecules of the present invention (or with expression or cloning vectors comprising them) and cultured in conventional nutrient media modified as appropriate for inducing viral production, inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In one embodiment, the host cell is an erythroid cell. In another embodiment, the erythroid cell is a human erythroid cell.
The culture conditions, such as media, temperature, pH, and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in M. Butler, Mammalian Cell Biotechnology: a Practical Approach, IRL Press (1991) and Sambrook et al., supra.
Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl2, CaPO4, liposome-mediated, and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Gene 23:315 (1983) and WO 89/05859. For mammalian cells without such cell walls, the calcium phosphate precipitation method described in Virology 52:456 457 (1978) can be employed. General aspects of mammalian cell host system transfections are described in U.S. Pat. No. 4,399,216. Transformations into yeast are typically carried out according to the method as described in J. Bact. 130:946 (1977) and Proc. Natl. Acad. Sci. (USA) 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see, for example, Methods in Enzymology 185:527 (1990) and Nature 336:348 (1988).
In various other aspects, the present invention provides an isolated nucleic acid molecule comprising an open reading frame or its complement thereof, wherein the open reading frame is comprised in SEQ ID NO: 1. In one embodiment, the open reading frame is comprised in SEQ ID NO: 128. In another embodiment, the open reading frame is comprised in SEQ ID NO: 129.
In some aspects, the present invention provides an isolated human erythrovirus having a genome comprising a nucleotide sequence as set forth in SEQ ID NO: 1. In one embodiment, the nucleotide sequence is as set forth in SEQ ID NO:128 or SEQ ID NO:129.
In other aspects, the present invention provides a kit. The kit can be developed using the nucleic acid sequences disclosed herein. These sequences can be used as primers in nucleic acid amplification reactions, and/or as probes in a nucleic acid hybridization method. The kits are useful for determining the presence of a parvovirus nucleic acid sequence in a sample. Components in the kit can either be obtained commercially or made according to well known methods in the art. In addition, the components of the kit can be in solution or lyophilized as appropriate. In one embodiment, the components are in the same compartment, and in another embodiment, the components are in separate compartments. In the preferred embodiment, the kit further comprises instructions for use.
The following examples are provided for illustration only.
EXAMPLES
Example 1
Detection of Parvovirus B19 D11 Variant by PCR
The D11 variant is detected from a biological source such as, for example, plasma, blood, bone marrow, or tissue sample for organ screening.
Approximately 1 ml of plasma or other biological source sample is added to approximately 200 μl of a buffered solution containing reagents designed to facilitate the precipitation of parvovirus B19 virions. To facilitate capsid disruption and pelleting, the buffered solution contains a chaotropic agent (e.g., guanidine isothiocyanate) and/or a detergent (e.g., sodium dodecyl sulfate (SDS)). Samples are mixed thoroughly by vortexing or inversion, and centrifuged. The supernatant is discarded and approximately 600 μl of a buffered solution containing a chaotropic agent such as, for example, guanidine thiocyanate is added to the pellet to completely disrupt the viral capsids. The parvovirus B19 DNA is then precipitated using, for example, approximately 700 μl isopropanol. Samples are thoroughly mixed by vortexing or inversion, and centrifuged using conditions that allow for the majority of the DNA to be recovered. Optionally, the pellet is washed, for example with approximately 1 ml 70% ethanol. After centrifugation, the parvovirus B19 variant DNA is recovered following resuspension of the pellet in a buffered solution to a volume (e.g., 200 μl) that allows for subsequent DNA amplification.
The D11 variant nucleic acid is amplified via PCR using techniques well-known in the art. For example, about 35 to 45 μl of a working master mix comprising of i) a buffer solution, ii) D 11 variant-specific oligonucleotides of which one is biotinylated on its 5′ end, iii) dideoxy-trinucleotides, and iv) DNA polymerase is added into a PCR tube or plate for amplification. Five to 15 μl of the resuspended sample is added to the PCR tube or plate containing the working master mix. Using a standard PCR thermocycler, such as the BioRad iCycler (Bio-Rad Laboratories), the samples and all relevant controls are subjected to PCR amplification. The PCR amplification condition comprises, for example, i) a step of denaturation (e.g. 94° C. for 60 seconds), ii) about forty cycles comprising thermal denaturation (e.g. 94° C. for 15 seconds) and polymerase extension (e.g. 65° C. for 30 seconds), and iii) a final step of polymerase extension (e.g. 60° C. for 7 minutes).
Following amplification, the resulting amplicons are detected using a variety of methods well known in the art. For example, the amplicons are detected by first denaturing the resulting amplified product and annealing the denatured nucleic acid sequences with complementary RNA probes to form RNA:DNA complexes. RNA probes are designed from the parvovirus B19 variant sequence. The complexes will be captured on streptavidin-coated plates and detected and/or quantified using, for example, antibodies directed to the complexes in an ELISA format.
There are a variety ELISA formats known in the art. For example, fifty μl of a sample diluent (e.g. Multiprep Specimen Diluent (MP DIL) (Roche Molecular Systems, Branchburg, N.J.)) is placed into a 96 well plate and 25 μl of a denaturation reagent (1.25 N NaOH) is added. Five to 10 μl of the amplified material is then added to each well containing the sample diluent and denaturation solution. After all the samples are transferred, the ELISA plates are covered and the samples mixed by placing the plates on a rotary shaker for about 30 to 60 seconds. The plates are incubated at room temperature for 10 to 20 minutes before adding about 25 μl of the RNA probe (500 pmole/L in neutralization buffer) to each well. After all the samples are transferred, the plates are covered and the samples mixed by placing the plates on a rotary shaker for about 30 to 60 seconds followed by incubation at 65° C. for 25 to 35 minutes.
After incubation, the contents of each well is transferred into another 96 well plate previously coated with streptavidin. After all the samples are transferred, the plate(s) are covered and the samples are mixed by placing the plates on a rotary shaker for 25 to 35 minutes. This latter step allows for the RNA:DNA complexes comprising the incorporated biotin label to bind to the streptavidin-coated plate. After the incubation and removal of the contents of the well, 100 μl of an appropriately diluted antibody coupled to alkaline phosphatase that is targeted to recognize the RNA:DNA complexes is added to each well and incubated at room temperature for about 25 to 35 minutes on a rotary shaker.
Following incubation, each well is washed about 4-5 times with a buffered wash solution (e.g. 80 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween 20) to remove non-binding reagents. The RNA:DNA:antibody complexes that are bound to the plate are detected using an alkaline phosphatase substrate such as, for example, p-nitrophenyl phosphate (pNPP). The rate of reaction or intensity of the signal is measured at 405 nanometers using a standard ELISA plate reader. The amount of color corresponds to the relative amount of the RNA:DNA:antibody complex present in the well. The quantity of the D11 variant is interpolated from a standard curve comprised of various concentrations of the D11 variant or suitable control standard (e.g. calibrated plasmids, amplicons, etc.) that are treated in the same manner as the samples to be determined.
Example 2
Detection of Parvovirus B19 D11 Variant by Real-Time PCR
The D11 variant is also detected and/or quantified using real-time PCR, which is well known in the art.
Samples comprising viral nucleic acids are prepared as described above. Amplification is carried out in a PCR tube or plate using an appropriate volume of PCR working master mix. The working master mix comprises a buffer solution comprising i) D11 variant specific oligonucleotides of which one contains a fluorescent dye on its 5′ end and a quencher near or at its 3′ end, ii) deoxyribonucleotides, and iii) a thermophilic DNA polymerase with 5′ to 3′ exonuclease activity. Five to 15 μL of the resuspended samples are added to the PCR tube or plate containing the working master mix for amplification. Using real-time PCR such as, for example, the AB7300 Real-Time PCR System (Applied Biosystems), the samples and all relevant controls are amplified.
Real-time PCR amplification conditions comprise, for example, i) a step of denaturation (e.g. 94° C. for 60 seconds), ii) about forty cycles comprising thermal denaturation (e.g. 94° C. for 15 seconds) and polymerase extension (e.g. 65° C. for 30 seconds), and iii) a final step of polymerase extension (e.g. 60° C. for 7 minutes).
During amplification, the resulting amplicons are detected using an appropriate energy source to excite the fluorescent dye cleaved from the oligonucleotide and filters to capture the fluorescence emitted. This detection is performed during each amplification cycle by measuring the relative fluorescence of the target and comparing it to the background fluorescence of the sample and instrument. This relative fluorescence is plotted against a standard curve derived using known amounts of a characterized molecular standard and a quantity is assigned to the sample based on the known quantity of the controls used to generate the standard curve.
Example 3
Parvovirus B19 D11 Variant Infectivity Assay
To determine infectivity of the D11 variant, primer(s) and a probe are used to amplify and detect an amplicon from the D11 variant mRNA. The amplicon serves as an indicator of viral replication as described in Virology 301:374-380 (2002).
Cultured cells susceptible to parvovirus B19 infection or replication are either infected with D11 variant or transfected with the viral DNA. Following viral gene expression, the cells are lysed by a suitable buffer such as, for example, the guanidine-based buffer described above. Nucleic acid is recovered from the lysate by alcohol-based precipitation or by binding to a matrix in a column (e.g., Promega SV Total RNA Isolation System) or particle format such as magnetic beads (e.g., Ambion's MagMAX Viral RNA Isolation Kits). The recovered nucleic acid are washed to remove cellular debris and resuspended in a suitable solution, such as water or TE buffer. A fraction of the recovered nucleic acid is treated with DNase to degrade DNA. Accordingly, two types of samples are prepared, one with total infected cell nucleic acid and one enriched for infected cell RNA.
Oligonucleotide primer or primers and an intervening probe comprising DNA sequences unique to the D11 variant described herein are used to amplify and detect D11 variant RNA in the samples. The viral RNA is reverse transcribed into cDNA using standard techniques well known in the art. For example, specific primers or random oligomers are used to prime the reverse transcription step using a reagent such as, for example, SuperScript III Reverse Transcriptase (Invitrogen).
Following reverse transcription of viral RNA into cDNA, the D11 variant nucleic acid is amplified using standard amplification technologies such as, for example, PCR, Transcription Mediated Amplification (TMA), or Ligase Chain Reaction. The amplified parvovirus B19 variant product is detected following hybridization to a labeled complementary oligonucleotide probe comprising a label such as, for example, a radioactive, a fluorescent, or a biotin label. The label, and hence the variant, is detected, for example, by direct detection of the label, by release of fluorescence during amplification in a TaqMan reaction, or through an indirect detection such as binding of an enzyme to the biotin or binding of a labeled antibody to the probe. Alternatively, one can target regions that result in spliced mRNAs. The shorter amplified mRNA target can be readily differentiated from native DNA template using methods that discriminate nucleic acids based on length.
Example 4
Detection of Parvovirus B19 D11 Variant without Prior Amplification
The D11 variant is detected and/or quantified using non-amplification techniques well known in the art. These methods allow detection of very low abundance nucleic acid sequences due to detection of single molecules with a high signal-to-noise ratio.
For example, the polymerase extension reaction, as described in Anal. Chem. 76:4169 (2004), is used to detect parvovirus B19 variants in samples prepared as described above. Highly fluorescent nucleic acid reporter molecules are made based on a unique sequence of the parvovirus B19 variant target nucleic acid as template. For example, the reporter molecule is made by annealing an oligonucleotide primer unique to the D11 variant to the target. The primer is then extended with a DNA polymerase and free oligonucleotides. One of these free oligonucleotides is labeled with a fluorophore. Fluorescence detection indicates the presence of the D11 variant in the sample.
D11 variant is also detected in a sample using confocal single molecule detection (SMD) of fluorescent molecular beacons (MB) as described in Analyst 130:483 (2005). This technique utilizes an oligonucleotide with sequences unique to the parvovirus B19 D11 variant described herein. In its native conformation, the molecular beacon forms a hairpin structure where the close proximity of the quencher and fluorophore prevents fluorescence. The hairpin structure will be released upon annealing to the target parvovirus B19 variant oligonucleotide so that the fluorophore is released from the quencher causing fluorescence. This increase in fluorescence is detected by confocal microscopy.
D11 variant is also detected in a sample using a method as described in Analyst 131:484 (2006). Such a technique allows for rapid and sensitive detection of parvovirus B19 variant nucleic acids using two-color quantum dots (QDs) and single-molecule coincidence detection. Quantum dots (QDs) have broad excitation spectra with a narrow emission bandwidth and possess exceptional photochemical stability with relatively high quantum yield. According to this method, two biotinylated oligonucleotide probes unique to the D11 variant are used to recognize and detect specific complementary target DNA through a sandwich hybridization reaction. The DNA hybrid is first captured on the surface of one type of QD through specific streptavidin-biotin binding. A different type of QD with a different emission spectrum binds the other end of the DNA hybrid to form a QD/DNA hybrid/QD complex. This hybrid possesses coincident spectra due to the binding of both QDs that is absent when target D11 variant DNA is not present.
Example 5
Differential Detection of Parvovirus B19 D11 Variant-Specific Sequences
The D11 variant described herein is also differentially detected from among other parvovirus B19 such as Au, A6, V9, and D91.1. Samples are prepared as described above and D11 variant nucleic acid is detected using nucleic acid oligonucleotides that specifically target variant sequence but not the other genotypes. Sequence-specific variations among parvovirus B19 genotypes is determined using various alignment algorithms and/or programs known in the art including, but not limited to FASTA, BLAST, or ENTREZ (available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.). FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.).
The targeted nucleic acid can be genomic DNA from intact virions or viral mRNA from parvovirus replicating in cells. Viral DNA or mRNA is prepared as described above and optionally, hybrid capture methods for the selection of specific sequences might be added in tandem to facilitate the enrichment of targeted nucleic acids. Nucleic acid sequences specific to the D11 variant are used as oligonucleotide primers for specific amplification of the variant nucleic acid, as labeled oligonucleotide probes for detection (e.g. incorporation of fluorophores, dyes, or other molecules used for detection), or as selective capture and retention moieties so as to enrich for the target variant while eliminating non-specific nucleic acid sequences.
Nucleic acid amplification and detection are performed as described above. For example, the variant nucleic acids are detected as part of an amplification reaction using labeled oligonucleotide probes (e.g. TaqMan or molecular beacons). Alternatively, variant nucleic acids are differentially amplified using primers specific to the D11 variant described herein and visualized using a variety of methods including, but not limited to gel electrophoresis, micro-well plate systems for detection of amplified nucleic acids, and various fluorogenic methodologies for generating signal as described in J. Virol Methods 136:210 (2006) and J. Clin. Microbiol. 44:2212 (2006).
Example 6
Identification of Conserved Regions of the Parvovirus B19 DNA Genome
To identify novel target regions of the parvovirus B19 genome that would expand the specificity of B19 detection assays to include detection of parvovirus B19 Genotypes 1, 2, 3, and variants and subtypes within each genotype, in silico analysis was performed to determine highly conserved regions of the parvovirus B19 genome by comparing Genotype 1, 2 and 3 partial and full coding sequences including the novel D11, E3, and P1 variants of the present invention.
A total of seven searches of GenBank (release 159) were performed to identify DNA sequences of parvovirus B19 variants and Genotypes 1, 2, and 3 prototype strains present in the database. These searches were performed using the following terms: ‘parvovirus B19’, ‘Human parvovirus B19’, ‘Human parvovirus A6’, ‘Human parvovirus V9’, ‘Human parvovirus D91.1’, ‘parvovirus B19 genotype’, and ‘parvovirus B19 variant’. The identified DNA sequences were imported into the local Vector NTI database and filtered to exclude sequences shorter than 100 bases or longer than 5600 bases, sequences representing human B19 receptor and immunoglobulin genes, circular vectors, parvovirus 4, and parvovirus of other species such as simian and canine. Sequences shorter than 100 bases were omitted since the flanking regions are most likely primer derived and not necessarily actual native parvovirus sequence. Sequences longer than 5600 bases were omitted because the parvovirus B19 genome is only 5600 bases and sequences longer than this includes flanking regions of vector that would alter the consensus sequence. The other omitted sequences are not representative of native parvovirus B19 sequence, and therefore should not be included in the overall consensus calculation. The remaining DNA sequences including the novel D11, E3, and P1 variants of the present invention were aligned using the AlignX program in Vector NTI, v7.1 according to the Alignment Settings in Table 1. These are the default settings in Vector NTI, v.7.1. Pairwise alignment settings dictate the alignment of two individual sequences to each other, while multiple alignment settings dictate how each of the individual pairwise alignments are aligned with all other pairwise alignments.







TABLE 1







Alignment Settings for pairwise alignment (left) and multiple


alignment (right) utilized to generate a multiple sequence


alignment in Vector NTI, v7.1








Pairwise Alignment
Multiple Alignment










Parameter
Setting
Parameter
Setting













Use FAST Algorithm
N/A
Gap Opening Penalty
15


K-tuple size
N/A
Gap Extension Penalty
6.66


Number of Best
N/A
Gap Separation Penalty Range
8


Diagonals


Window Size
N/A
No End Gap Separation
N/A




Penalty


Gap Penalty
N/A
% Identity for Alignment
40




Delay


Gap Opening Penalty
15
Residue Specific Gaps Off
N/A


Gap Extension Penalty
6.66
Hydrophilic Residue Gap Off
N/A




Transition Weighting
0









Consensus sequences were calculated at 100%, 99%, 98%, 97%, and 95% by Vector NTI according to the following Alignment Display Settings: Consider only identical residues in consensus calculation, Ignore gaps in consensus calculation, and the residue fraction for consensus was set at 1.0, 0.99, 0.98, 0.97, and 0.95 respectively. The function of using a specific sequence as a consensus was left un-checked. Table 2 shows the similarity table used in the consensus calculation. Only strong similarities were considered in consensus calculation.







TABLE 2







Alignment display setup nucleotide similarity table










Residue
Strong Similarity







A
A



C
C



G
G



T
T










The consensus sequence calculated at 97% provided the highest degree of sequence homology and minimal nucleotide gaps between the filtered parvovirus sequences. This was the consensus sequence used to design oligonucleotide primer and probe sets for evaluation in real-time PCR detection of parvovirus B19.
Initial identification of primer and probe sets was performed by visual inspection of conserved regions for presence of target regions. A target region was defined as a region in which primers and detection probes could be designed. Target regions were identified that had between 100 and 200 nucleotides and have two flanking sequences (primer binding regions) of 15 to 25 continuous nucleotides with an internal continuous sequence (probe binding region) greater than 20 nucleotides. Also, the internal sequence had to be in close proximity (within 20 nucleotides) to either flanking sequence. The identified primer sequences were further evaluated for primer dimer formation, presence of secondary structure, and melting temperature. Identified detection probes were evaluated to ensure; absence of secondary structure, absence of primer/probe dimers, absence of a 5′ guanine residue, and the melting temperature was 7 to 10° C. higher than the flanking primer pair.
The combination of search terms in GenBank identified 881 DNA sequences that were imported into the local Vector NTI database. The 881 imported sequences were filtered according to the criteria stated above and the remaining 565 DNA sequences were aligned using the AlignX program in Vector NTI, v7.1.
A DNA consensus sequence was generated from the alignment of 565 parvovirus B19 DNA sequences utilizing the Alignment Display Settings of Vector NTI shown in Tables 1 and 2. Different DNA consensus sequences were generated with 100%, 99%, 98%, 97%, and 95% homology by changing the ‘Residue fraction for consensus’ value while retaining all other settings. The 97% consensus sequence was aligned with the Au isolate of B19 (Accession number: M13178) as the reference sequence (FIG. 5) to show the locations of the conserved regions and where no nucleotides are present at the 97% level of sequence homology.
A review of the 97% DNA sequence homology consensus revealed two regions of the parvovirus B19 genome as highly conserved across all of the 565 aligned parvovirus B19 sequences. Relative to parvovirus B19 Genotype 1 prototype strain Au, these two regions encompass nucleotides 1351 to 2426 (FIG. 6) and nucleotides 3704 to 4804 (FIG. 7).
The highly conserved sequence spanning nucleotides 1351 through 2426 was analyzed further to locate any DNA regions that could be utilized as target regions. Four (4) target regions were identified and designated region 1, 2, 4 and 5 (FIG. 8 and Table 3).







TABLE 3







Target Region and oligonucleotide sequences derived from highly


conserved regions of the parvovirus B19 genome.















SEQ




Target


ID




Region
Function
Name
NO:
Sequence (5′ to 3′)
Tm





1
Forward
B19_2043F
136
TGAAACCCCGCGCTCTA
59.6



primer






 



Reverse
B19_2171R
137
AACTAACAGTTCACGAAACTG
56.7



primer






 



Detection
B19_2069F_FAM
138
TCCCCGGGACCAGTTCAGGAGAA
68.1



probe






 


2
Forward
B19_1962F
139
TCAGCAGCAGTGGTGGT
59.6



primer






 



Reverse
B19_2043R
140
TAGAGCGCGGGGTTTCA
59.6



primer






 



Detection
B19_1979F-FAM
141
TGAAAGCTCTGAAGAACTCAGTGAAAGCAGCTTT
67.0



probe






 



Forward
B19_1903F
142
AATGCAGATGCCCTCCAC
59.9



primer






 



Detection
B19_1962F-FAM
143
TCAGCAGCAGTGGTGGTGAAAGCTCTGAA
68.9



probe






 



Reverse
B19_2027R
144
TGTTCCAGGCGCCTG
58.9



primer






 


3
Forward
B19_4700F
145
CACAGCTACAGATGCAAA
55.3



primer






 



Reverse
B19_4769R
146
GGTGCACACGGCTTTT
56.7



primer






 



Detection
B19_4733R_FA
147
TGTCCACAATTCTTCAGGCTTTTCATATCC
64.6



probe
M





 



Detection
B19_4733F_FAM
148
TGGATATGAAAAGCCTGAAGTATTGTGGAC
64.6



probe






 



Forward
B19_4672F
149
GGTCATTTACCATATGTACT
54.2



primer






 



Detection
B19_4703F-FAM
150
AGCTACAGATGCAAANCAACACCACAGACA†
66.7



probe






 


4
Forward
B19_1500F
151
GAAAACTTTCCATTTAATGATGT
53.8



primer






 



Reverse
B19_1631R
152
ATTTTTTGATCTACCCTGGT
54.2



primer






 



Detection
B19_1537F-FAM
153
TTGGTGGTCTGGGATGAAGG
62.4



probe






 


5
Forward
B19_1411F
154
GTTTTATGGGCCGCCAAGTA
60.4



primer






 



Reverse
B19_1537R
155
TTCATCCCAGACCACCAAGG
62.4



primer






 



Detection
B19_1450F-FAM
156
ATGGCTATTGCTAAAACTGTTCCAGTGTA
63.2



probe






 



Detection
B19_1492F-FAM
157
TGGAATAATGAAAACTTTCCATTTAATGATGTAG
61.0



probe






 



Detection
B19_1448F-FAM
158
CAATGGCCATTGCTAAAAGTGTTCCA
63.0



probe





†N = a universal base analogue including, without limitation, 5-nitroindole, inosine, and 4-nitrobenzimidazole.






The highly conserved sequence spanning nucleotides 3704 through 4804 was also analyzed further to locate any potential target regions. One (1) target region was identified and designated as region 3 (FIG. 8 and Table 3).
Regions 1, 2, 4 and 5 are positioned within the parvovirus B19 gene coding for non-structural protein 1 (NS 1) and region 3 is positioned within overlapping genes coding for viral protein 1 and 2 (VP1, VP2) (FIG. 8). FIG. 9 shows alignment of e.g., primer/probe sequences of target region 5 to parvovirus B19 Genotype 1 prototype strain Au (i.e., SEQ ID NO:130).
Accordingly, an alignment of 565 parvovirus B19 variant and Genotype 1, 2, and 3 DNA sequences identified five highly conserved target regions of the parvovirus B19 genome at 97% DNA sequence homology. At the 97% DNA sequence homology, 13 oligonucleotide primers in 5 different regions were designed with 100 percent homology to the consensus sequence. At the same level, 10 oligonucleotide detection probes were designed. Of these 10 probes, 6 have a 100 percent homology. At least one probe (i.e., B19—4703-FAM) has 97% homology to the 97% consensus sequence. This probe, B19—4703-FAM, has one base at nucleotide 16 from the 5′ end that is blank in the consensus sequence. A universal base (5-nitroindole) was inserted in this position to facilitate hybridization of the probe to the B19 viral DNA sequence. Exemplary universal base analogues include, without limitation, 5-nitroindole, inosine, and 4-nitrobenzimidazole.
Example 7
Evaluating a New Targeting Strategy for Parvovirus B19 Detection
The consensus sequence resulting from alignment of full and partial genomic sequences of human parvovirus B19 Genotypes 1, 2, and 3 present in GenBank was utilized to identify highly conserved regions of the parvovirus B19 genome and design oligonucleotide primers and probes within each conserved region (see Table 3 and FIG. 10). To evaluate experimentally whether the conserved DNA sequence regions identified as potential target regions for parvovirus B19 actually enhance the specificity of detection of parvovirus B19 variants and genotypes, oligonucleotide probes and primers were designed for amplification and detection of the identified conserved regions. A total of 16 primer/probe combination sets were evaluated (Table 4).







TABLE 4







Primer/Probe Combinations for B19 New Targeting


Strategy Evaluation.












Primer/
SEQ ID

Tm


Region
Probe Set
NO:
Oligonucleotide Name
(° C.)





1
1
136
B19_2043F
59.6




137
B19_2171R
56.7




138
B19_2069F_FAM
68.1


2
2a
139
B19_1962F
59.6




140
B19_2043R
59.6




141
B19_1979F-FAM
67.0



2b
139
B19_1962F
59.6




144
B19_2027R
58.9




141
B19_1979F-FAM
67.0



2c
142
B19_1903F
59.9




144
B19_2027R
58.9




141
B19_1979F-FAM
67.0



2d
142
B19_1903F
59.9




140
B19_2043R
59.6




141
B19_1979F-FAM
67.0



2e
142
B19_1903F
59.9




140
B19_2043R
59.6




143
B19_1962F-FAM
68.9



2f
142
B19_1903F
59.9




144
B19_2027R
58.9




143
B19_1962F-FAM
68.9


3
3a
145
B19_4700F
55.3




146
B19_4769R
56.7




148
B19_4733F_FAM
64.6



3b
145
B19_4700F
55.3




146
B19_4769R
56.7




147
B19_4733R_FAM
64.6



3c
149
B19_4672F
54.2




146
B19_4769R
56.7




150
B19_4703F-FAM
66.7



3d
149
B19_4672F
54.2




146
B19_4769R
56.7




148
B19_4733F_FAM
64.6



3e
149
B19_4672F
54.2




146
B19_4769R
56.7




147
B19_4733R_FAM
64.6


4
4
151
B19_1500F
53.8




152
B19_1631R
54.2




153
B19_1537F-FAM
62.4


5
5a
154
B19_1411F
60.4




155
B19_1537R
62.4




156
B19_1450E-FAM
63.2



5b
154
B19_1411F
60.4




155
B19_1537R
62.4




157
B19_1492F-FAM
61.0



5c
154
B19_1411F
60.4




155
B19_1537R
62.4




158
B19_1448F-FAM
63.0









All oligonucleotides were purchased from Integrated DNA Technologies (IDT) (Coralville, Iowa) with 100 nmole synthesis scale for primers and 250 nmole scale for probes. Each oligonucleotide was suspended in RNase-, DNase-free water. A B19 master mix was prepared with the oligonucleotide sets (Table 4). The plasma samples utilized for extraction panels are: NAT-056 (Genotype 1), E3 (i.e., SEQ ID NO: 128) (Genotype 1), P1 (i.e., SEQ ID NO: 129) (Genotype 3). Plasma containing parvovirus B19 Genotype 2 is currently not available for assessment. A full-length Genotype 2 amplicon may be obtained for future assessment. Dilution panels of P1 (i.e., SEQ ID NO: 129) and E3 (i.e., SEQ ID NO: 128) were made for initial extraction by 10 fold serial dilutions of the stock material to a 10−9 final dilution in normal human plasma. The 10−4, 10−7, and 10−9 test sample dilutions were evaluated with the primer and probe combinations in Table 4. Each extraction set included the test samples and controls listed in Table 5.







TABLE 5







Test samples and controls utilized for assessment of primersand probes











Sample
Dilution
N Size







B19 Negative
NA
3



Control



B19 Low Positive
NA
3



Control



B19 High Positive
NA
3



Control



B19 100 IU/mL
NA
2



E3 1.0 × 10−4
1:104
2



E3 1.0 × 10−7
1:107
2



E3 1.0 × 10−9
1:109
2



P1 1.0 × 10−4
1:104
2



P1 1.0 × 10−7
1:107
2



P1 1.0 × 10−9
1:109
2










Amplification and detection were performed on the AB7300 Real Time PCR System (Applied Biosystems, Foster City, Calif.). All extracted samples were amplified in duplicate. The amplification controls included five B19 quantification standards for each oligonucleotide set. The assay controls consisted of the B19 Negative Control, B19 Low Positive Control, and B19 High Positive Control. The test master mixes used to amplify the extracted samples were made using the different primer and probe combinations listed in Table 4. Each primer and probe combination was amplified at 2 anneal/extension temperatures: 60° C. and 55° C. to accommodate lower melting temperatures (Tm) of some primers and probes (Table 4).
Results for parvovirus B19 detection for each of the primer and probe combinations at 55° C. and 60° C. for each sample concentration is shown in Table 6.







TABLE 6







Summary of parvovirus B19 amplification and detection for primer/probe


sets amplified at 55° C. and 60° C. anneal/extension temperatures.










Primer/Probe

Temperature












Set
Sample
55° C.
60° C.
Comments





1
P1
+
+
Good detection



E3
+
+


2a
P1
+
+
Did not detect 10−9 test sample dilution at 55° C.






All test sample dilutions were detected at 60° C.



E3
+
+


2b
P1
−
−
Primer/probe failure likely due to proximity of



E3
−
−
primer to probe proximity


2c
P1
−
−
Reverse primer B19_2027R unable to amplify






and detect target



E3
−
−


2d
P1
+
+
Good detection, especially at 60° C.



E3
+
+


2e
P1
+
+
Good detection at 60° C.



E3
+
+
Did not detect all 10−9 test sample dilutions


2f
P1
−
−
Not analyzed since reverse primer shared



E3
−
−
with Primer/probe Set 2c


3a
P1
+
+
At 60° C., only 10−4 test sample dilution






amplified and detected



E3
+
+
Good detection at 55° C.


3b
P1
+
+
Good detection



E3
+
+


3c
P1
+
+
Good detection at 55° C.



E3
+
+
At 60° C., only 10−4 test sample dilution amplified






and detected


3d
P1
+
+
No robust amplification and detection at low






test sample concentrations



E3
+
+


3e
P1
+
+
No robust amplification and detection at low






test sample concentrations



E3
+
+


4
P1
+
−
Poor detection at 55° C.



E3
+
−
No detection at 60° C.


5a
P1
+
−



E3
+
−


5b
P1
+
+
Good detection



E3
+
+
Good detection


5c
P1
−
−



E3
+
+
Good detection





Key:


(+) indicates detection and


(−) indicates no detection.






Thirteen out of 16 primer/probe sets representing all 5 conserved regions showed detection of P1 (i.e., SEQ ID NO: 129) and/or E3 (i.e., SEQ ID NO: 128). Primer/probe Set 2b did not demonstrate amplification and detection. Further oligonucleotide and B19 genomic region sequence analysis proved that the probe and forward primer were positioned too close to each other (probe overlapping the 3′ end of the primer) to generate amplification and detection. Primer/probe Set 2c also did not demonstrate amplification and detection, most likely due to poor design/performance of the reverse primer. Primer/probe Set 2f was not analyzed because it shared the same reverse primer with Primer/probe Set 2c. The average CT values from the amplification and detection of P1 (i.e., SEQ ID NO: 129) and E3 (i.e., SEQ ID NO: 128) at both anneal/extension temperatures for 10 of the 12 primer/probe sets are shown in FIG. 11. Examples of amplification plots are shown for Primer/probe Sets 2d and 3b in FIGS. 12 and 13.
All 5 target regions that were identified as conserved regions of the parvovirus B19 genome showed detection of both P1 and E3 variants, confirming that these target regions were conserved at least for parvovirus B19 genotypes 1 and 3. Primer/probe sets designed to detect the 5 target regions provided good robust amplification and detection.

## Claims

1. A method for detecting a parvovirus B19 nucleic acid in a sample, the method comprising:
a) performing PCR with at least a portion of the sample using a forward primer, the nucleotide sequence of which consists of SEQ ID NO:145, and a reverse primer, the nucleotide sequence of which consists of SEQ ID NO:146; and
b) determining the presence or absence of an amplicon, wherein the presence of the amplicon is indicative of the presence of the parvovirus B19 nucleic acid in the sample, wherein determining comprises annealing an oligonucleotide to the amplicon.

2. The method of claim 1, wherein the oligonucleotide comprises the sequence set forth in:
(SEQ ID NO:147) or the sequence fully complementary thereto.

3. The method of claim 1, wherein the presence of the amplicon indicates the presence of a parvovirus B19 genotype 1 or 3 if present in the sample.