Abstract:
Highly specific oligonucleotide primers and probes useful in a rapid and specific method for detecting the presence of Group B Streptococcal (GBS) or  Streptococcus agalactiae  infection in a biological sample.

Description:
FIELD OF THE INVENTION  
         [0001]    This invention relates to methods for detecting Group B streptococci (GBS) infections, particularly to methods allowing a rapid and accurate diagnosis to prevent and treat neonatal GBS infections.  
         BACKGROUND  
         [0002]    Group B streptococci (GBS) are responsible for a broad range of severe human diseases, predominantly the life-threatening bacterial infections in neonates and very young infants. Approximately 70 to 80% of infant infections occur in the first few days of life, so-called early-onset disease, while late-onset infections occur in infants between 1 week and 3 months of age. Newborns with early-onset GBS disease usually acquire the organism during delivery from their GBS-colonized mothers, manifesting in sepsis and meningitis which cause not only illness and death, but long term disabilities such as hearing loss, impaired vision, developmental problems, and cerebral palsy.  
           [0003]    In order to substantially reduce the incidence of early-onset GBS disease, prenatal screening for GBS and intrapartum antimicrobial prophylaxis are now highly recommended in the United States. However, since these strategies require the frequent use of antibiotics, antibiotic resistant GBS or other bacterial agents might emerge during the perinatal period. In addition, these measures are unlikely to prevent late-onset infections, prematurity, and stillbirths related to GBS, while obviously not addressing GBS disease in nonpregnant adults. GBS are increasingly recognized as a frequent cause of invasive infections in pregnant women and clinically ill and older adults, such as those suffering from diabetes, cirrhosis, malignancies and immunodifciencies.  
           [0004]    Currently, culture, including broth culture in selective medium, is the gold standard method for detection of GBS. However, the culture methods require up to 36 hours to obtain results and predict only 87% of women likely to be colonized by GBS at delivery. A rapid, sensitive, and specific test for detection of GBS directly from clinical specimens would allow for a simpler and more efficient prevention program.  
           [0005]    Rapid tests have been developed, such as the rapid antigen-based tests, but these tests are neither sensitive nor specific enough to substitute for bacterial culture. The most widely used hybridization-based test to date is the Accuprobe Group B Streptococcocus Identification Test™ (Gen-Probe, San Diego, Calif.). GBS-specific polymerase chain reaction (PCR) assays using real-time PCR have been described which provide improved detection of GBS (Bergeron et al. (2000) N. Engl. J. Med. 343, 175-179).  
         BRIEF SUMMARY OF THE INVENTION  
         [0006]    The present invention features a rapid and accurate PCR-based assay for  Streptococcus agalactiae,  the organism responsible for neonatal Group B Streptococcal (GBS) infections.  
           [0007]    The method of the invention presented here included a pair of hybridization primers (SEQ ID NO: 1 and 2) specific to the portion of the cfb gene (FIG. 1; SEQ ID NO: 3) between positions 328 and 451 (SEQ ID NO:4) encoding the CAMP factor. The CAMP factor is a diffusible extracellular protein and is produced by the majority of GBS. The gene encoding CAMP factor, cfb gene (GenBank access number: X72754), is present in virtually every GBS isolate and has been used for the development of a PCR based identification of GBS (Danbing K. et al., 2000, Clinical Chemistry, 46, 324-331).  
           [0008]    Further, the instant invention also provides a specific probe (SEQ ID NO: 5) designed to recognize the sequence amplified between the primers, e.g., the amplicons of the cfb gene comprised of the 123 bp sequence of SEQ ID NO:4, allowing real-time detection by using fluorescence measurements.  
           [0009]    The present invention also includes a pair of GBS specific PCR amplification primers (SEQ ID NO: 6 and 7) specific for a portion of the sip gene (FIG. 2; SEQ ID NO:8) between positions 778 and 857 (SEQ ID NO:9). GBS sip gene (GenBank access number: AF151357, AF151358, AF151359, AF151360, AF151361, AF151362) encodes a 53-kDa protein called surface immunogenic protein (“Sip”), which is present in all serotypes of GBS. Further included is a specific probe (SEQ ID NO: 10) recognizing the amplicons allowing real-time detection by using fluorescence measurement.  
           [0010]    Accordingly, in a first aspect, the invention features a method of testing for the presence of  Streptococcus agalactiae,  comprising:  
           [0011]    (a) hybridization a nucleic acid sample suspected of being infected with Group B Streptococcal (GBS), with (i) a first pair of CAMP-based Group B Streptococcal (GBS)-specific primers, and (ii) a second pair of Sip-based GBS-specific primers, under conditions wherein GBS-related nucleic acids are amplified; and  
           [0012]    (b) detecting the presence of GBS-related nucleic acids.  
           [0013]    In one embodiment, the first pair of CAMP-based GBS primers are the oligonucleotides of SEQ ID NOs:1 and 2. In another embodiment, the second pair of Sip-based GBS primers are the oligonucleotides of SEQ ID NOs:5 and 6. In a further embodiment, the first pair of CAMP-based GBS primers are the oligonucleotides of SEQ ID NOs:1 and 2 and the second pair of Sip-based GBS primers are the oligonucleotides of SEQ ID NOs:5 and 6.  
           [0014]    In one embodiment, the method of the invention is used in conjunction with SYGR as a means of amplicon detection. SYGR is a fluorescent dye which binds to double stranded DNA and fluoresces strongly when bound to double stranded DNA. In another embodiment, step (a) is conducted in the presence of the probe of SEQ ID NOs:5. In another embodiment, step (a) is conducted in the presence of the probe of SEQ ID NO:10. In a more specific embodiment, step (a) is conducted in the presence of the pro be of SEQ ID NOs:5. In another embodiment, step (a) is conducted in the presence of the probes of SEQ ID NOs:5 and 10.  
           [0015]    The probes of SEQ ID NOs: 5 and 10 are double labeled with a fluorophore at the 5′ and a quencher at the 3′, so when the probe is intact the flourophore is not able to fluorescence (TaqMan®Probe, IDT, Coralville, Iowa). During PCR extension, the probes hybridized to the amplicons is cleaved by the 5′-3′ exonuclease activity of Taq polymerase, resulting releasing of the 5′ fluorophore from its quencher. The intensity of the fluorescence increases as more amplicons being synthesized.  
           [0016]    In a further embodiment, the nucleic acid sample is a biological sample obtained from a patient to be tested for the presence of GBS. DNA from any biological sample extracted by any known method may be used in the method of the invention. Biological samples include, for example, vaginal or anal specimens, amniotic fluid, spinal fluid, or plasma.  
           [0017]    In a further embodiment, step (a) in conducted in a volume of 0.2-100 μl; in further embodiments, step the reaction is conducted in a volume of less than 50 μl, or less than 25 μl; in a still further embodiment, the reaction is conducted in less than 15 μl.  
           [0018]    In a second aspect, the invention features a method of diagnosing a Group B Streptococcal (GBS) infection, comprising:  
           [0019]    (a) contacting a GBS-related target nucleic acid with (i) a first pair of CAMP-based Group B Streptococcal (GBS)-specific primers, (ii) a CAMP-based Group B Streptococcal (GBS)-specific probe, (iii) a second pair of Sip-based GBS-specific primers, and (iv) a Sip-based GBS-specific probe, under conditions wherein GBS-related nucleic acids are amplified; and  
           [0020]    (b) detecting the amplified products, wherein detection of amplified products indicates the presence of a GBS infection.  
           [0021]    In a third aspect, the invention features an in vitro method for detecting the presence of  Streptococcus agalactiae  in a biological sample, comprising:  
           [0022]    (a) releasing nucleic acids from said biological sample;  
           [0023]    (b) performing PCR in a total volume of between 0.2-100 μl in the presence of a first pair of primers comprising SEQ ID NOs: 1 and 2, and a second pair of primers comprising SEQ ID NOs:6 and 7, and labeled probes comprising SEQ ID NOs:5 and 10, under conditions wherein the presence of a  Streptococcus agalactiae -related nucleic acid sequence results in an amplified and labeled PCR product; and  
           [0024]    (c) detecting the presence of PCR product with either specific probes or SYBG.  
           [0025]    In a fourth aspect, the invention features a method detecting a Group B Streptococcal (GBS) infection in a patient, comprising:  
           [0026]    (a) obtaining a biological sample from the patient;  
           [0027]    (b) releasing nucleic acids from said biological sample;  
           [0028]    (c) performing PCR in a total volume of between 0.2-100 μl in the presence of a first pair of primers comprising SEQ ID NOs:1 and 2, and a second pair of primers comprising SEQ ID NOs:6 and 7, and labeled probes comprising SEQ ID NOs:5 and  10, under conditions wherein the presence of a    Streptococcus agalactiae -related nucleic acid sequence results in an amplified and labeled PCR product; and  
           [0029]    (d) detecting the presence of PCR product with specific fluorescent labeled probes or SYBG, wherein the presence of degraded probe indicates the presence of a GBS infection.  
           [0030]    Other objects and advantages will become apparent from a review of the ensuing detailed description taken in conjunction with the following illustrative drawing. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0031]    [0031]FIG. 1 is the cfb gene (SEQ ID NO: 3) showing primers (SEQ ID NO: 1 and 2) specific to the portion of the cJb gene between positions 328 and 451 (SEQ ID NO:4) encoding the CAMP factor, and a specific probe (SEQ ID NO: 5) designed to recognize the sequence amplified between the primers comprised of the 123 bp sequence of SEQ ID NO:4.  
         [0032]    [0032]FIG. 2 is the sip gene (SEQ ID NO:8) with the positions of the primers (SEQ ID NO: 6 and 7) specific for a portion of the sip gene between positions 778 and 857 (SEQ ID NO:9) and a specific probe (SEQ ID NO: 10) recognizing the amplicons. 
     
    
     DETAILED DESCRIPTION  
       [0033]    Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular methods, compositions, and experimental conditions described, as such methods and compounds may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only the appended claims.  
         [0034]    As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus for example, references to “the method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.  
         [0035]    Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and described the methods and/or materials in connection with which the publications are cited.  
       DESCRIPTION OF THE INVENTION  
       [0036]    The method described herein is a rapid and accurate screening test for GBS that can be performed at the time of delivery and which obviates the need for prenatal screening and reduce the use of antibiotic prophylaxis in women who are not colonized.  
         [0037]    Recently, Bergeron et al. (2000) N. Engl. J. Med. 343, 175-179, described a rapid PCR technique capable of correctly identifying more than 90% women colonized with GBS. Both the sensitivity and specificity of this technique appeared to be in the range that would be acceptable for clinical use.  
         [0038]    The method described in the instant specification uses a standard PCR machine Further, the probes and primers of the instant invention provide a very high and specific sensitivity for rapid detection of GBS.  
         [0039]    Accordingly, the present invention resides in part in a process for amplifying two specific nucleic acid sequences present in a nucleic acid or mixture thereof, using two pairs of specific primers for polymerization, and two specific probes for detecting the amplified sequences. Further, the present invention provides an important advantage in allowing quick detection of the presence of the GBS pathogen so that appropriate medical intervention is available to the infection patient(s) more quickly.  
       EXAMPLES  
       [0040]    The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.  
       Example 1  
     Primer and Probe Design  
       [0041]    The sequences of cfb and sip genes are obtained from GenBank. The primers and probes were designed with the aid of Primer Express 1.0 (PE Applied Biosystem). The possible homologies of the primers and probes with other none GBS genes were checked using NCBI Blast program and Megaline (DNA Star Lasergene).  
         [0042]    Camp based GBS-specific primers:  
                                   Forward primer:   5′ GATGTATCTATCTGGAACTCTAGTG 3′;   (SEQ ID NO:1)                   Reverse primer:   5′ GGCTTGATTATTACTATTTACATGATTTACCA 3′;   (SEQ ID NO:2)               Probe:   5′ F-AGAAGTACATGCTGATCAAGTGACAACTCCACA-Q 3′.   (SEQ ID NO:5)          
 
         [0043]    Sip Based GBS-Specific Primers:  
                                                                                                                         Forward primer:   (SEQ ID NO:6)                    5′ GTGCATCACCAGAGCATGTAT 3′;                                Reverse primer″   (SEQ ID NO:7)                    5′ CGCTTGTAACTTACTGTCTGTAGCTG 3′;                                Probe:   (SEQ ID NO:10)                    5′ F-AGCTCCAGCAGTTCCTGTGACTACGACTT 3′.              
 
         [0044]    The specificity of the primers and probes was tested with real-time PCR (Taqman assay) using genomic DNAs isolated from the following organisms (listed in Table 1): nine GBS serotypes (serotype Ia, Ib, Ic, II, III, IV, V, VI and VII; American Type Culture Collection and National Center for Streptococcus, Canada); 10 clinical GBS isolates; 60 clinical samples; a wide variety of gram-positive and gram-negative bacterial strains as well as two yeast strains and HSV type 1 and 2.  
                           TABLE 1                                   PATHOGEN   TYPE                             Pseudomonas aeruginosa     Gram − Bacteria             Proteus mirabilis     Gram − Bacteria             Klebsiella oxytoca     Gram − Bacteria             Klebsiella pneumoniae     Gram − Bacteria             Escherichia coli  (clinical isolate 1)   Gram − Bacteria             Escherichia coli  (clinical isolate 2)   Gram − Bacteria             Acinetobacter baumannii     Gram − Bacteria             Serratia marcescens     Gram − Bacteria             Enterobacter aerogenes     Gram + Bacteria             Enterococcus faecium     Gram + Bacteria             Staphylococcus aureus  (clinical isolate 1)   Gram + Bacteria             Staphylococcus aureus  (clinical isolate 2)   Gram + Bacteria             Streptococcus pyo genes     Gram + Bacteria             Streptococcus viridans     Gram + Bacteria             Listeria monocytogenes     Gram + Bacteria             Enterococcus sps.     Gram + Bacteria             Candida glabrata     Yeast             Candida albicans     Yeast             Streptococcus Group C     Gram + Bacteria             Streptococcus Group G     Gram + Bacteria             Streptococcus Group F     Gram + Bacteria             Enterococcus faecalis     Gram + Bacteria             Streptococcus pneumoniae     Gram + Bacteria             Staphylococcus epidermidis (C−)     Gram + Bacteria             Gardenerella vaginalis     Gram + Bacteria             Micrococcus sps.     Gram + Bacteria             Haemophilus influenzae     Gram − Bacteria             Neisseria gonorrhoeae     Gram − Bacteria             Moraxella catarrahlis     Gram − Bacteria             Salmonella sps.     Gram − Bacteria             Chlamydia trachomatis     Gram − Bacteria             Peptostreptococcus productus     Gram + Bacteria             Peptostreptococcus anaerobius     Gram + Bacteria             Lactobacillus fermentum     Gram + Bacteria             Eubacterium lentum     Gram + Bacteria           Herpes Simplex Virus I (HSV I)   Virus           Herpes Simplex Virus II (HSV II)   Virus                      
 
         [0045]    Assay procedure. A typical PCR was conducted with the GBS specific probes and primers of the invention under the following conditions:  
         [0046]    20 mM Tris-HCl, pH 8.4  
         [0047]    50 mM KCl  
         [0048]    4 mM MgCl2  
         [0049]    0.2 mM dNTPs  
         [0050]    400 μM primers (SEQ ID NOs:1, 2, 4, 5)  
         [0051]    200 μM probes (SEQ ID NOs:3, 6)  
         [0052]    10 fg to 1 ng DNA  
         [0053]    1-2 U of Taq polymerase  
         [0054]    Total volume is 15 μl and the reaction is carried out in a LightCycler with: 25 sec denaturing at 94° C.; followed by 50 cycles of 94° C. for 3 sec., and 60° C. for 20 sec.  
         [0055]    Results. Both sets of primers and probes recognized all the nine GBS serotypes, the 10 clinical isolates, and the clinical samples, which are GBS positive by culturing method. There are no cross reactivities with any of the other pathogens.  
     
       
       
         1 
         
           
             10  
           
           
             1  
             25  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            1 

gatgtatcta tctggaactc tagtg                                           25 

 
           
             2  
             32  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            2 

tggtaaatca tgtaaatagt aataatcaag cc                                   32 

 
           
             3  
             1467  
             DNA  
             Streptococcus agalactiae  
           
            3 

atattggtaa gaagaaattt ccttaaaaat aagattaaat aggttgtaaa gtatccgtat     60 

gggttttact tgaaaaacta aattaaatta tcaagaaatt accccccagg ataggcgcca    120 

agaatattat acccacttga taatggtaag ttttatgcta aaaatgcagt ttacttgtaa    180 

taatgttaaa tataggggga aagaaagcgc tttgacgacc ttttggacaa gtagtaagat    240 

accaacatgg gccctgtaaa ttaaaaatac tgcagtagaa gtgattttag tttaaaggag    300 

gaaatttatt atgaacgtta cacatatgat gtatctatct ggaactctag tggctggtgc    360 

attgttattt tcaccagctg tattagaagt acatgctgat caagtgacaa ctccacaagt    420 

ggtaaatcat gtaaatagta ataatcaagc ccagcaaatg gctcaaaagc ttgatcaaga    480 

tagcattcag ttgagaaata tcaaagataa tgttcaggga acagattatg aaaaaccggt    540 

taatgaggct attactagcg tggaaaaatt aaagacttca ttgcgtgcca accctgagac    600 

agtttatgat ttgaattcta ttggtagtcg tgtagaagcc ttaacagatg tgattgaagc    660 

aatcactttt tcaactcaac atttaacaaa taaggttagt caagcaaata ttgatatggg    720 

atttgggata actaagctag ttattcgcat tttagatcca tttgcttcag ttgattcaat    780 

taaagctcaa gttaacgatg taaaggcatt agaacaaaaa gttttaactt atcctgattt    840 

aaaaccaact gatagagcta ccatctatac aaaatcaaaa cttgataagg aaatctggaa    900 

tacacgcttt actagagata aaaaagtact taacgtcaaa gaatttaaag tttacaatac    960 

tttaaataaa gcaatcacac atgctgttgg agttcagttg aatccaaatg ttacggtaca   1020 

acaagttgat caagagattg taacattaca agcagcactt caaacagcat taaaataata   1080 

tttgtatttt tcgtgtgatg ctgtcgactt cgtgattttg tactaccatg attgttatga   1140 

ttaaaagatt tacgacaata gtcataatag tagaacgatg tcaccatttt aaataataaa   1200 

gtgattagtc atttgactaa atttgccaag tatcaaagga aataaagatt atgactaaaa   1260 

agataactgt tgtagcatta gaaacattga ttgcccagca taataatatc catttgatag   1320 

acgttcgtga agagcatgag tatcgtggag ggcatattcc aggtgcgata aatcttcctt   1380 

tgagtcactc agtcataagt ttgaacagtt agataaaata aggaatatta tcttgttggc   1440 

aacgaggggg aagatctatt agagcat                                       1467 

 
           
             4  
             124  
             DNA  
             Streptococcus agalactiae  
           
            4 

gatgtatcta tctggaactc tagtggctgg tgcattgtta ttttcaccag ctgtattaga     60 

agtacatgct gatcaagtga caactccaca agtggtaaat catgtaaata gtaataatca    120 

agcc                                                                 124 

 
           
             5  
             33  
             DNA  
             Artificial Sequence  
             
               probe  
             
           
            5 

agaagtacat gctgatcaag tgacaactcc aca                                  33 

 
           
             6  
             21  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            6 

gtgcatcacc agagcatgta t                                               21 

 
           
             7  
             26  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            7 

cagctacaga cagtaagtta caagcg                                          26 

 
           
             8  
             1301  
             DNA  
             Streptococcus agalactiae  
           
            8 

atgaaaatga ataaaaaggt actattgaca tcgacaatgg cagcttcgct attatcagtc     60 

gcaagtgttc aagcacaaga aacagatacg acgtggacag cacgtactgt ttcagaggta    120 

aaggctgatt tggtaaagca agacaataaa tcatcatata ctgtgaaata tggtgataca    180 

ctaagcgtta tttcagaagc aatgtcaatt gatatgaatg tcttagcaaa aattaataac    240 

attgcagata tcaatcttat ttatcctgag acaacactga cagtaactta cgatcagaag    300 

agtcatactg ccacttcaat gaaaatagaa acaccagcaa caaatgctgc tggtcaaaca    360 

acagctactg tggatttgaa aaccaatcaa gtttctgttg cagaccaaaa agtttctctc    420 

aatacaattt cggaaggtat gacaccagaa gcagcaacaa cgattgtttc gccaatgaag    480 

acatattctt ctgcgccagc tttgaaatca aaagaagtat tagcacaaga gcaagctgtt    540 

agtcaagcag cagctaatga acaggtatca acagctcctg tgaagtcgat tacttcagaa    600 

gttccagcag ctaaagagga agttaaacca actcagacgt cagtcagtca acaacagtat    660 

caccagcttc tgttgccgct gaaacaccag ctccagtagc taaagtagca ccggtaagaa    720 

ctgtagcagc ccctagagtg gcaagtgtta aagtagtcac tcctaaagta gaaactggtg    780 

catcaccaga gcatgtatca gctccagcag ttcctgtgac tacgacttca acagctacag    840 

acagtaagtt acaagcgact gaagttaaga gcgttccggt agcacaaaaa gctccaacag    900 

caacaccggt agcacaacca gcttcaacaa caaatgcagt agctgcacat cctgaaaatg    960 

cagggctcca acctcatgtt gcagcttata aagaaaaagt agcgtcaact tatggagtta   1020 

atgaattcag tacataccgt gcaggtgatc caggtgatca tggtaaaggt ttagcagtcg   1080 

actttattgt aggtaaaaac caagcacttg gtaatgaagt tgcacagtac tctacacaaa   1140 

atatggcagc aaataacatt tcatatgtta tctggcaaca aaagttttac tcaaatacaa   1200 

atagtattta tggacctgct aatacttgga atgcaatgcc agatcgtggt ggcgttactg   1260 

ccaaccatta tgaccatgtt cacgtatcat ttaacaaata a                       1301 

 
           
             9  
             80  
             DNA  
             Streptococcus agalactiae  
           
            9 

gtgcatcacc agagcatgta tcagctccag cagttcctgt gactacgact tcaacagcta     60 

cagacagtaa gttacaagcg                                                 80 

 
           
             10  
             29  
             DNA  
             Artificial Sequence  
             
               probe  
             
           
            10 

agctccagca gttcctgtga ctacgactt                                       29