# Patent

## Title
Detection of nucleic acids by type-specific hybrid capture method

## Abstract

Target-specific hybrid capture (TSHC) provides a nucleic acid detection method that is not only rapid and sensitive, but is also highly specific and capable of discriminating highly homologous nucleic acid target sequences. The method produces DNA-RNA hybrids which can be detected by a variety of methods.

## Background
The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license to others on reasonable terms as provided by the terms of Grant/Contract No. N44 AI085335 awarded by the National Institutes of Health.


FIELD OF INVENTION
This invention relates to the field of nucleic acid detection methods in general and more particularly relates to the detection of nucleic acids by target-specific hybrid capture method.
BACKGROUND OF THE INVENTION
The detection of specific nucleic acid sequences present in a biological sample is important for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility to disease, and measuring response to various types of treatment. A common technique for detecting and quantitating specific nucleic acid sequences is nucleic acid hybridization.
Various hybridization methods are available for the detection and study of nucleic acids. In a traditional hybridization method, the nucleic acids to be identified are either in a solution or affixed to a solid carrier. The nucleic acids are detected using labelled nucleic acid probes which are capable of hybridizing to the nucleic acids. Recently, new hybridization methods have been developed to increase the sensitivity and specificity of detection. One example is the hybrid capture method described in U.S. application Ser. No. 07/792,585. Although these new hybridization methods offer significant improvements over the traditional methods, they still lack the ability to fully discriminate between highly homologous nucleic acid sequences.
It is therefore an object of the present invention to provide a hybridization method which is not only rapid and sensitive, but is also highly specific and capable of discriminating highly homologous nucleic acid target sequences.
SUMMARY OF THE INVENTION
The present invention provides a novel nucleic acid detection method, referred to herein as target-specific hybrid capture (“TSHC”). TSHC is a highly specific and sensitive method which is capable of discriminating and detecting highly homologous nucleic acid target sequences.
In one embodiment, the method relates to detecting a target nucleic acid wherein the targeted nucleic acid is hybridized simultaneously, or sequentially, to a capture sequence probe and an unlabelled signal sequence probe. These probes hybridize to non-overlapping regions of the target nucleic acid and not to each other so that double-stranded hybrids are formed. The hybrids are captured onto a solid phase and detected. In a preferred embodiment, an DNA-RNA hybrid is formed between the target nucleic acid and the signal sequence probe. Using this method, detection may be accomplished, for example, by binding a labeled antibody capable of recognizing an DNA-RNA hybrid to the double-stranded hybrid, thereby detecting the hybrid.
In another embodiment, the signal sequence probe used in the detection method is a nucleic acid molecule which comprises a DNA-RNA duplex and a single stranded nucleic acid sequence which is capable of hybridizing to the target nucleic acid. Detection may be accomplished, for example, by binding a labeled antibody capable of recognizing the DNA-RNA duplex portion of the signal sequence probe, thereby detecting the hybrid formed between the target nucleic acid, the capture sequence probe and the signal sequence probe.
In yet another embodiment, the signal sequence probe used in the detection method is a molecule which does not contain sequences that are capable of hybridizing to the target nucleic acid. Bridge probes comprising sequences that are capable of hybridizing to the target nucleic acid as well as sequences that are capable of hybridizing to the signal sequence probe are used. In this embodiment, the signal sequence probe comprises a DNA-RNA duplex portion and a single stranded DNA sequence portion containing sequences complementary to sequences within the bridge probe. The bridge probe, which hybridizes to both the target nucleic acid and the signal sequence probe, therefore serves as an intermediate for connecting the signal sequence probe to the target nucleic acid and the capture sequence probe hybridized to the target nucleic acid.
In another embodiment of the TSHC method of the invention, blocker probes comprising oligonucleotides complementary to the capture sequence probes are used in the method to eliminate excess capture sequence probe, thereby reducing the background signal in detection and increasing specificity of the assay.
The present invention also relates to novel probes. These probes are nucleic acid sequences which can function in various hybridization assays, including, for example, the TSHC assay.



BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a schematic diagram illustrating one embodiment of the target-specific hybrid capture method.
FIG. 2 is a schematic diagram illustrating one embodiment of the target-specific hybrid capture method.
FIG. 3 is a schematic diagram illustrating possible mechanisms of action of an embodiment that employs fused capture sequence probes in target-specific hybrid capture detection.
FIG. 4 shows the analytical sensitivity and specificity of target-specific hybrid capture detection of HSV-1.
FIG. 5 shows the analytical sensitivity and specificity of target-specific hybrid capture detection of HSV-2.
FIGS. 6A-6D show the various embodiments of the target-specific hybrid capture-plus method.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for detecting the presence of nucleic acids in test samples. More specifically, the invention provides a highly specific and sensitive method which is capable of discriminating and detecting highly homologous nucleic acid sequences.
Any source of nucleic acid, in purified or non-purified form, can be utilized as the test sample. For example, the test sample may be a food or agricultural product, or a human or veterinary clinical specimen. Typically, the test sample is a biological fluid such as urine, blood, plasma, serum, sputum or the like. Alternatively the test sample may be a tissue specimen suspected of carrying a nucleic acid of interest. The target nucleic acid in the test sample may be present initially as a discrete molecule so that the sequence to be detected constitutes the entire nucleic acid, or may only be a component of a larger molecule. It is not necessary that the nucleic acid sequence to be detected be present initially in a pure form. The test sample may contain a complex mixture of nucleic acids, of which the target nucleic acid may correspond to a gene of interest contained in total human genomic DNA or RNA or a portion of the nucleic acid sequence of a pathogenic organism which organism is a minor component of a clinical sample.
The target nucleic acid in a test sample can be DNA or RNA, such as messenger RNA, from any source, including bacteria, yeast, viruses, and the cells or tissues of higher organisms such as plants or animals. Methods for the extraction and/or purification of such nucleic acids are well known in the art. Target nucleic acids may be double-stranded or single-stranded. In the present method, it is preferred that the target nucleic acids are single-stranded or made single-stranded by conventional denaturation techniques prior to the hybridization steps of the method. In a preferred embodiment, base denaturation technique is used to denature the double-stranded target DNA.
The term “oligonucleotide” as the term is used herein refers to a nucleic acid molecule comprised of two or more deoxyribonucleotides or ribonucleotides. A desired oligonucleotide may be prepared by any suitable method, such as purification from a naturally occurring nucleic acid, by molecular biological means, or by de novo synthesis. Examples of oligonucleotides are nucleic acid probes described herein.
Nucleic acid probes are detectable nucleic acid sequences that hybridize to complementary RNA or DNA sequences in a test sample. Detection of the probe indicates the presence of a particular nucleic acid sequence in the test sample. In one embodiment, the target-specific hybrid capture method employs two types of nucleic acid probes: capture sequence probe (CSP) and signal sequence probe (SSP). A capture sequence probe comprises a nucleic acid sequence which is capable of hybridizing to unique region(s) within a target nucleic acid and being captured onto a solid phase. A signal sequence probe comprises a nucleic acid sequence which is capable of hybridizing to regions within a target nucleic acid that are adjacent to the unique regions recognized by the CSP. The sequences of CSP and SSP are selected so that they would not hybridize to the same region of a target nucleic acid or to each other.
In addition, the CSP and the SSP are selected to hybridize to regions of the target within 50,000 bases of each other. The distance between the sequence to which the CSP hybridizes within the target nucleic acid and the sequence to which the SSP hybridizes is preferably between 1 to 50,000 bases, more preferably, the distance is less than 3,000 bases. Most preferably, the distance is less than 1,000 bases.
The CSP used in the detection method can be DNA, RNA, peptide nucleic acids (PNAs) or other nucleic acid analogues. PNAs are oligonucleotides in which the sugar-phosphate backbone is replaced with a polyamide or “pseudopeptide” backbone. In a preferred embodiment, the CSP is DNA. The CSP has a minimum length of 8 bases, preferably between 15 to 100 bases long, and more preferably between 20 to 40 bases long. The CSP is substantially complementary to the sequence within a target nucleic acid to which it hybridizes. The sequence of a CSP is preferably at least 75% complementary to the target hybridization region, more preferably, 100% complementary to this sequence. It is also preferred that the CSP contains less than or equal to 75% sequence identity, more preferably less than 50% sequence identity, to non-desired sequences believed to be present in a test sample. The sequence within a target nucleic acid to which a CSP binds is preferably 12 bases long, more preferably 20-40 bases long. It may also be preferred that the sequences to which the CSP hybridizes are unique sequences or group-specific sequences. Group-specific sequences are multiple related sequences that form discrete groups.
In one embodiment, the CSP used in the detection method may contain one or more modifications in the nucleic acid which allows specific capture of the probe onto a solid phase. For example, the CSP may be modified by tagging it with at least one ligand by methods well-known to those skilled in the art including, for example, nick-translation, chemical or photochemical incorporation. In addition, the CSP may be tagged at multiple positions with one or multiple types of labels. For example, the CSP may be tagged with biotin, which binds to streptavidin; or digoxigenin, which binds to anti-digoxigenin; or 2,4-dinitrophenol (DNP), which binds to anti-DNP. Fluorogens can also be used to modify the probes. Examples of fluorogens include fluorescein and derivatives, phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, Texas Red or other proprietary fluorogens. The fluorogens are generally attached by chemical modification and bind to a fluorogen-specific antibody, such as anti-fluorescein. It will be understood by those skilled in the art that the CSP can also be tagged by incorporation of a modified base containing any chemical group recognizable by specific antibodies. Other tags and methods of tagging nucleotide sequences for capture onto a solid phase coated with substrate are well known to those skilled in the art. A review of nucleic acid labels can be found in the article by Landegren, et al., “DNA Diagnostics-Molecular Techniques and Automation”, Science, 242: 229-237 (1988), which is incorporated herein by reference. In one preferred embodiment, the CSP is tagged with biotin on both the 5′ and the 3′ ends of the nucleotide sequence. In another embodiment, the CSP is not modified but is captured on a solid matrix by virtue of sequences contained in the CSP capable of hybridization to the matrix.
The SSP used in the detection method may be a DNA or RNA. In one particular embodiment of the invention, the SSP and target nucleic acid form a DNA-RNA hybrid. Therefore, in this embodiment, if the target nucleic acid is a DNA, then the preferred SSP is an RNA. Similarly, if the target nucleic acid is RNA, then the preferred SSP is a DNA. The SSP is generally at least 15 bases long. However, the SSP may be up to or greater than 1000 bases long. Longer SSPs are preferred. The SSP may comprise a single nucleic acid fragment, or multiple smaller nucleic acid fragments each of which is preferably between 15 to 100 bases in length.
In another embodiment, the SSP used in the detection method comprises a DNA-RNA duplex and a single stranded nucleic acid sequence capable of hybridizing to the target nucleic acid (FIG. 6A). The SSP may be prepared by first cloning a single stranded DNA sequence complementary to sequences within the target nucleic acid into a single-stranded DNA vector, then hybridizing RNA complementary to the DNA vector sequence to generate a DNA-RNA duplex. For example, if M13 is used as the DNA vector, M13 RNA is hybridized to the M13 DNA sequence in the vector to generate a DNA-RNA duplex. The resulting SSP contains a DNA-RNA duplex portion as well as a single stranded portion capable of hybridizing to sequences within the target nucleic acid. The single stranded DNA should be at least 10 bases long, and may be up to or greater than 1000 bases long. Alternatively, the DNA-RNA duplex portion of the SSP may be formed during or after the reaction in which the single stranded portion of the SSP is hybridized to the target nucleic acid. The SSP can be linear, circular, or a combination of two or more forms. The DNA-RNA duplex portion of the SSP provides amplified signals for the detection of captured hybrids using anti-DNA-RNA antibodies as described herein.
In yet another embodiment, the SSP used in the detection method is a molecule which does not contain sequences that are capable of hybridizing to the target nucleic acid. In this embodiment, bridge probes comprising sequences capable of hybridizing to the target nucleic acid as well as sequences capable of hybridizing to the SSP are used. The bridge probes can be DNA, RNA, peptide nucleic acids (PNAs) or other nucleic acid analogues. In one embodiment (FIG. 6B), the SSP comprises a DNA-RNA duplex portion and a single stranded portion containing sequences complementary to sequences within the bridge probe. The bridge probe, which is capable of hybridizing to both the target nucleic acid and the SSP, therefore serves as an intermediate for connecting the SSP to the target nucleic acid and the CSP hybridized to the target nucleic acid. The SSP may be prepared as described above. In another embodiment (FIG. 6C), the SSP used in the detection method comprises multiple sets of repeat sequences as well as a single stranded RNA sequence capable of hybridizing to the bridge probe. A DNA oligonucleotide probe containing sequences complementary to the repeat sequences may be used to hybridize to the SSP to generate the RNA-DNA duplex needed for signal amplification. In yet another embodiment (FIG. 6D), the bridge probe contains a poly(A) tail in addition to sequences which are capable of hybridizing to the target nucleic acid. The SSP used in this example comprises poly(dT) DNA sequences. The bridge probe therefore is capable of hybridizing to the SSP via its poly(A) tail. A RNA probe comprising poly(A) sequences may be used to hybridize to the remaining poly(dT) DNA sequences within SSP to form a RNA-DNA duplex. The SSP comprising poly(dT) sequences and the RNA probe comprising poly(A) sequences are preferably 100 to 5,000 bases long.
The SSP used in the detection method of the invention can be unmodified, or modified as with the CSP using methods described above and/or known in the art. In a preferred embodiment, the SSP is a covalently unmodified probe.
It is understood that multiple CSPs and/or SSPs can be employed in the detection method of the invention.
In another embodiment, an oligonucleotide probe comprising complementary sequences of two or more distinct regions of the target nucleic acid are fused together and used as the capture sequence probe in the method of the invention. Alternatively a single probe can be designed and produced which contains sequences complementary to single or multiple target nucleic acids. This type of probe is also referred to herein as a “fused” CSP. As shown in Example 5, the fused capture sequence probe works as effectively as the combination of two unfused CSPs when used at the same concentration.
The nucleic acid probes of the invention may be produced by any suitable method known in the art, including for example, by chemical synthesis, isolation from a naturally-occurring source, recombinant production and asymmetric PCR (McCabe, 1990 In: PCR Protocols: A guide to methods and applications. San Diego, Calif., Academic Press, 76-83). It may be preferred to chemically synthesize the probes in one or more segments and subsequently link the segments. Several chemical synthesis methods are described by Narang et al. (1979 Meth. Enzymol. 68: 90), Brown et al. (1979 Meth. Enzymol. 68: 109) and Caruthers et al. (1985 Meth. Enzymol. 154: 287), which are incorporated herein by reference. Alternatively, cloning methods may provide a convenient nucleic acid fragment which can be isolated for use as a promoter primer. A double-stranded DNA probe is first rendered single-stranded using, for example, conventional denaturation methods prior to hybridization to the target nucleic acids.
Hybridization is conducted under standard hybridization conditions well-known to those skilled in the art. Reaction conditions for hybridization of a probe to a nucleic acid sequence vary from probe to probe, depending on factors such as probe length, the number of G and C nucleotides in the sequence, and the composition of the buffer utilized in the hybridization reaction. Moderately stringent hybridization conditions are generally understood by those skilled in the art as conditions approximately 25° C. below the melting temperature of a perfectly base-paired double stranded DNA. Higher specificity is generally achieved by employing incubation conditions having higher temperatures, in other words more stringent conditions. Chapter 11 of the well-known laboratory manual of Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, second edition, Cold Spring Harbor Laboratory Press, New York (1990) (which is incorporated by reference herein), describes hybridization conditions for oligonucleotide probes in great detail, including a description of the factors involved and the level of stringency necessary to guarantee hybridization with specificity. Hybridization is typically performed in a buffered aqueous solution, for which conditions such as temperature, salt concentration, and pH are selected to provide sufficient stringency such that the probes hybridize specifically to their respective target nucleic acid sequences but not any other sequence.
Generally, the efficiency of hybridization between probe and target improve under conditions where the amount of probe added is in molar excess to the template, preferably a 2 to 106 molar excess, more preferably 103 to 106 molar excess. The concentration of each CSP provided for efficient capture is at least 25 fmoles/ml (25 pM) in the final hybridization solution, preferably between 25 fmoles to 104 fmoles/ml (10 nM). The concentration of each SSP is at least 15 ng/ml in the final hybridization solution, preferably 150 ng/ml. Table A shows the conversion of SSP concentrations expressed in ng/ml to molar basis.







TABLE A







Conversion of SSP Concentration From ng/ml to fmoles/ml









SSP Concentration
SSP Concentration in fmoles/ml (pM)










in ng/ml
SSP is a 3 kb RNA
SSP is a 5 kb RNA












 15 ng/ml
15.1
9


150 ng/ml
151
90


600 ng/ml
606
364









Hybridization of the CSP and the SSP to the target nucleic acid may be performed simultaneously or sequentially and in either order. In one embodiment, hybridization of the CSP and hybridization of the SSP to the target nucleic acid are performed simultaneously. The hybrid formed is then captured onto a solid phase coated with a substrate to which ligand attached to the CSP binds with specificity. In another embodiment, hybridization of the SSP to the target nucleic acid is performed after the hybridization of the CSP to the target nucleic acid. In this case, the CSP may be immobilized on a solid phase before or after hybridization. In this embodiment, both the CSP and the target may be bound to the solid phase during the SSP hybridization reaction.
It will be understood by those skilled in the art that a solid phase or matrix includes, for example, polystyrene, polyethylene, polypropylene, polycarbonate or any solid plastic material in the shape of plates, slides, dishes, beads, particles, cups, strands, chips and strips. A solid phase also includes glass beads, glass test tubes and any other appropriate glass product. A functionalized solid phase such as plastic or glass that has been modified so that the surface contains carboxyl, amino, hydrazide, aldehyde groups, nucleic acid or nucleotide derivatives can also be used. Any solid phase such as plastic or glass microparticles, beads, strips, test tubes, slides, strands, chips or microtiter plates can be used.
In one preferred embodiment, the CSP is labelled with biotin, and streptavidin-coated or avidin-coated solid phase is employed to capture the hybrid. More preferably, streptavidin-coated microtiter plates are used. These plates may be coated passively or covalently.
The captured hybrid may be detected by conventional means well-known in the art, such as with a labelled polyclonal or monoclonal antibody specific for the hybrid, an antibody specific for one or more ligands attached to the SSP, a labelled antibody, or a detectable modification on the SSP itself.
One preferred method detects the captured hybrid by using an anti-RNA-DNA antibody. In this embodiment, the anti-RNA-DNA antibody is preferably labelled with an enzyme, a fluorescent molecule or a biotin-avidin conjugate and is non-radioactive. The label can be detected directly or indirectly by conventional means known in the art such as a colorimeter, a luminometer, or a fluorescence detector. One preferred label is, for example, alkaline phosphatase. Other labels known to one skilled in the art can also be employed as a means of detecting the bound double-stranded hybrid.
Detection of captured hybrid is preferably achieved by binding the conjugated antibody to the hybrid during an incubation step. Surfaces are then washed to remove any excess conjugate. These techniques are known in the art. For example, manual washes may be performed using either an EppendorfTM Repeat Pipettor with a 50 ml CombitipTM (Eppendorf, Hamburg, Germany), a Corning repeat syringe (Corning, Corning, N.Y.), a simple pump regulated by a variostat, or by gravity flow from a reservoir with attached tubing. Commercially available tube washing systems available from Source Scientific Systems (Garden Grove, Calif.) can also be used.
Bound conjugate is subsequently detected by a method conventionally used in the art, for example, colorimetry or chemiluminescence as described at Coutlee, et al., J. Clin. Microbiol. 27: 1002-1007 (1989). Preferably, bound alkaline phosphatase conjugate is detected by chemiluminescence by adding a substrate which can be activated by alkaline phosphatase. Chemiluminescent substrates that are activated by alkaline phosphatase are well known in the art.
In another embodiment, the target specific hybrid capture method of the invention employs blocker probes in addition to the CSP and SSP. A blocker probe comprises sequences that are complementary to the sequences of the CSP. The sequence of a blocker probe is preferably at least 75% complementary to the sequence of the CSP, more preferably, 100% complementary to the CSP. The addition of the blocker probes to the hybridization reaction mixture prevents non-hybridized CSP from hybridizing to cross-reactive nucleic acid sequences present in the target and therefore increases the specificity of the detection.
The blocker probe is generally at least 5 bases long, preferably 12 bases long. The concentration of the blocker probe in the hybridization reaction is preferably in excess to that of the CSP and SSP. Preferably, the blocker probe is present in a 2-fold molar excess, although, it may be present in an up to 10,000-fold molar excess. The blocker probes can be DNA, RNA, peptide nucleic acids (PNAs) or other nucleic acid analogues.
In one embodiment, blocker probes complementary to the full-length or near full-length of the CSP are used. Following the reaction in which the hybrid between CSP, SSP and the target nucleic acid is formed, one or more blocker probes may be added to the reaction and the hybridization is continued for a desired time. The hybridization products are then detected as described above.
In another embodiment, blocker probes complementary to only a portion of the CSP and are shorter than the CSP are used. These blocker probes have a lower melting temperature than that of the CSP. Preferably, the melting temperature of the blocker probe is 10 degrees lower than that of the CSP. In this case, the blocker probe is preferably added to the target nucleic acids simultaneously with the CSP and the SSP. Since the blocker probe has a lower melting temperature than the CSP, the initial temperature for hybridization is chosen such that the blocker probe does not interfere with the hybridization of the CSP to its target sequences. However, when the temperature of the hybridization mixtures is adjusted below the temperature used for target hybridization, the blocker probe hybridizes to the CSP and effectively blocks the CSP from hybridizing to cross-reactive nucleic acid sequences. For example, when the hybridization products are incubated at room temperature on a streptavidin-coated microtiter plate during hybrid capture, the blocker probes may be added.
The following examples illustrate use of the present amplification method and detection assay and kit. These examples are offered by way of illustration, and are not intended to limit the scope of the invention in any manner. All references described herein are expressly incorporated in toto by reference.
EXAMPLE 1
Target-Specific Hybrid Capture (TSHC) Assay Protocol
Herpes Simplex Virus 1 (HSV-1) and Herpes Simplex Virus 2 (HSV-2) viral particles of known concentration (Advanced Biotechnologies, Inc., Columbia, Md.) or clinical samples were diluted using either Negative Control Media (Digene Corp., Gaithersburg, Md.) or Negative Cervical Specimens (Digene). Various dilutions were made and aliquoted into individual microfuge tubes. A half volume of the Denaturation Reagent 5100-0431 (Digene) was added. Test samples were incubated at 65° C. for 45 minutes for denaturation of nucleic acids in the samples.
Following denaturation, a hybridization solution containing signal sequence probes (SSPs) (600 ng/ml each) and capture sequence probes (CSPs) (2.5 pmoles/ml each) was added to the sample, and incubated at 74° C. for 1 hour. Blocker probes in a solution containing one volume of 4× Probe Diluent (Digene), one volume of Denaturation Reagent and two volumes of the Negative Control Media were then added to the hybridization mixture and incubated at 74° C. for 15 minutes.
In a second series of experiments, following denaturation of nucleic acids, a hybridization mixture containing SSPs (600 ng/ml each), CSPs (2.5 pmoles/ml each), and blocker probes (250 pmoles/ml each) was added to the samples and incubated for one hour at 74° C.
Tubes containing reaction mixtures were cooled at room temperature for 5 minutes, and aliquots were taken from each tube and transferred to individual wells of a 96-well streptavidin capture plate (Digene). The plates were shaken at 1100 rpms for 1 hour at room temperature. The supernatants were then decanted and the plates were washed twice with SNM wash buffer (Digene) and inverted briefly to remove residual wash buffer. The alkaline-phosphatase anti-RNA/DNA antibody DR-1 (Digene) was then added to each well and incubated 30 minutes at room temperature. The wells were then subjected to multiple wash steps which include: 1) three washes with Sharp wash buffer (Digene) at room temperature; 2) incubation of the plate with the Sharp wash buffer for 10 minutes at 60° C. on a heat block; 3) two washes with the Sharp wash buffer at room temperature; and 4) one wash with the SNM wash buffer (Digene) at room temperature. Following removal of the residual liquid, luminescent substrate 5100-0350 (Digene) was added to each well and incubated for 15 minutes at room temperature. The individual wells were then read on a plate luminometer to obtain the relative light unit (RLU) signal.
Solutions containing Negative Control Media or known HSV Negative Cervical Specimens were used as negative controls for the test samples. The signal to noise ratio (S/N) was calculated as the ratio of the average RLU obtained from a test sample to the average RLU of the negative control. The signal to noise ratio was used as the basis for determining capture efficiency and the detection of target nucleic acids. A S/N value of 2 or greater was arbitrarily assigned as a positive signal while a S/N values less than 2 was considered negative. The coefficient of variation (CV) which is a determination of the variability of the experiment within one sample set was calculated by taking the standard deviation of the replicates, dividing them by the average and multiplying that value by 100 to give a percent value.
The capture sequence probes and the blocker probes used in experiments described in Examples 2-13 were synthesized using the method described by Cook et al. (1988 Nucl. Acid. Res., 16: 4077-95). Unless otherwise noted, the capture sequence probes used in the experiments described herein were labeled with biotins at their 5′ and 3′ ends.
The signal sequence probes used in experiments described in Examples 2-13 are RNA probes. These probes were prepared using the method described by Yisraeli et al. (1989, Methods in Enzymol., 180: 42-50).
EXAMPLE 2
The following tables describe the various probes used in experiments described in Examples 3-13.







TABLE 1







HSV-1 Clones from which HSV-1 Probes are derived











Clone
Host
Cloning
Insert
Sequence Location


Name
Vector
Site(s)
Size (bp)
within HSV-1





RH3
Dgx3
Hind III,
5720
39850-45570




Eco RI


R10
Blue
Eco RI
4072
64134-68206



Script SK+


RH5B
Blue
Eco RV,
4987
105108-110095



Script SK+
Eco RI


H19
Blue
Hind III
4890
133467-138349



Script SK+
















TABLE 2







HSV-2 Clones from which HSV-2 Probes are derived











Clone
Host
Cloning
Insert
Sequence Location


Name
Vector
Site(s)
Size (bp)
in HSV-2





E4A
Blue
Bam HI
3683
23230-26914



Script SK+


E4B
Blue
Bam HI
5600
26914-32267



Script SK+
Eco RI


I8
Blue
Hind III
2844
41624-44474



Script SK+


EI8
Dgx3
Hind III,
3715
44474-48189




Eco RI


4L
Blue
Bam HI,
4313
86199-90512



Script SK+
Eco RI
















TABLE 3







Capture Sequence Probes for HSV-1












Size
Location within


Probe
Sequence
(bp)
HSV-1













TS-1
(TTATTATTA)CGTTCATGTCGGCAAACAGCT
24
105040-105063



CGT(TTATTATTA) [SEQ ID NO:1]


TS-2
(TTATTATTA)CGTCCTGGATGGCGATACGGC
21
110316-110336



(TTATTATTA) [SEQ ID NO:2]


VH-3
CGTCCTGGATGGCGATACGGC
21
110316-110336



[SEQ ID NO:3]


NC-1
CGTTCATGTCGGCAAACAGCTCGT
24
105040-105063



[SEQ ID NO:4]


VH-4
CGTTCATGTCGGCAAACAGCTCGT-
45
105040-105063;


(fusion of
CGTCCTGGATGGCGATACGGC

110316-110336


VH3, NC-1)
[SEQ ID NO:5]


HZ-1
GATGGGGTTATTTTTCCTAAGATGGGGC
34
133061-133094



GGGTCC [SEQ ID NO:6]


VH-2
TACCCCGATCATCAGTTATCCTTAAGGT
28
138367-138394



[SEQ ID NO:7]


FD-1
AAACCGTTCCATGACCGGA [SEQ ID NO:8]
19
39281-39299


RA-2
ATCGCGTGTTCCAGAGACAGGC
22
39156-39177



[SEQ ID NO:9]


NC-2
CAACGCCCAAAATAATA [SEQ ID NO:10]
17
46337-46353


FD-2
GTCCCCGAaCCGATCTAGCG (note small
20
45483-45502



cap a is mutated base) [SEQ ID NO:11]


RA-4
CGAACCATAAACCATTCCCCAT
22
46361-46382



[SEQ ID NO:12]


ON-3
CACGCCCGTGGTTCTGGAATTCGAC
25
64105-64129



[SEQ ID NO:13]


HZ-2
(TTTATTA)GATGGGGTTATTTTTCCTAAGATG
34
133061-133094



GGGCGGGTCC [SEQ ID NO:14]


ZD-1
GGTTATTTTTCCTAAG [SEQ ID NO:15]
16
133064-133079


ZD-2
(ATTATT)GGTTATTTTTCCTAAG(ATTATT)
16
133064-133079



[SEQ ID NO:16]


F6R
ACGACGCCCTTGACTCCGATTCGTCATCGGAT
40
87111-87150



GACTCCCT [SEQ ID NO:17]


BRH19
ATGCGCCAGTGTATCAATCAGCTGTTTCGGGT
32
133223-133254



[SEQ ID NO:18]


F15R
CAAAACGTCCTGGAGACGGGTGAGTGTCGGC
38
141311-141348



GAGGACG [SEQ ID NO:19]


VH-1
GTCCCCGACCCGATCTAGCG [SEQ ID NO:20]
20
45483-45502


ON-4
GCAGACTGCGCCAGGAACGAGTA
23
68404-68426



[SEQ ID NO:21]


PZ-1
GTGCCCACGCCCGTGGTTCTGGAATTCGACAG
35
64105-64139



CGA [SEQ ID NO:22]


PZ-2
GCAGACTGCGCCAGGAACGAGTAGTTGGAGT
35
68404-68438



ACTG [SEQ ID NO:23]


FG-2
AAGAGGTCCATTGGGTGGGGTTGATACGGGA
36
105069-105104



AAGAC [SEQ ID NO:24]


FG-3
CGTAATGCGGCGGTGCAGACTCCCCTG
27
110620-110646



[SEQ ID NO:25]


FG-4
CCAACTACCCCGATCATCAGTTATCCTT
39
138362-138400



AAGGTCTCTTG [SEQ ID NO:26]


Hsv1-LF15R
(AAAAAAAAA)CAAAACGTCCTGGAGACGGGT
38
141311-141348


(SH-3)
GAGTGTCGGCGAGGACG [SEQ ID NO:27]


Hsv1-F15-2B
CAAAACGTCCTGGAGACGGGTGAGTGTCGGC
38
141311-141348


(GZ-1)
GAGGACG [SEQ ID NO:28]


Hsv1-F15-3B
CAAAACGTCC-bio-U-GGAGACGGGTGAG
38
141311-141348


(GZ-2)
TG-bio-U-CGGCGAGGACG [SEQ ID NO:29]





*Sequences in parentheses are “tail” sequences not directed at HSV.













TABLE 4







Blocker Probes for HSV-1












Size
Capture Probe to


Probe
Sequence
(bp)
which it hybridizes





EA-1
AGGAAAAATAACCCCATC [SEQ ID NO:30]
18
HZ-1


EA-2
GACCCGCCCCATCTT [SEQ ID NO:31]
15
HZ-1


ZD-3
GGACCCGCCCCATCTTAGGAAAAATAAC
34
HZ-1



CCCATC [SEQ ID NO:32]


NG-7
AAAAATAACCCCA [SEQ ID NO:33]
13
HZ-1


NG-8
CGCCCCATCTT [SEQ ID NO:34]
11
HZ-1


NG-4
CCATCTTAGGAAAAA [SEQ ID NO:35]
15
HZ-1


GP-1
ATAACTGATGATCGG [SEQ ID NO:36]
15
VH-Z


EA-3
CCACCCAATGGACCTC [SEQ ID NO:37]
16
FG-2


EA-4
GTCTTTCCCGTATCAACC [SEQ ID NO:38]
18
FG-2


EB-7
CGCCGCATTACG [SEQ ID NO:39]
12
FG-3


EB-8
AGGGGAGTCTGC [SEQ ID NO:40]
12
FG-3


GP-3
CTGTTTGCCGACA [SEQ ID NO:41]
13
VH-4


GP-4
TATCGCCATCCAG [SEQ ID NO:42]
13
VH-4


EB-9
ATGATCGGGGTAGT [SEQ ID NO:43]
14
FG-4


EB-10
AGAGACCTTAAGGATA [SEQ ID NO:44]
16
FG-4


NG-1
ATTCCAGAACCACGG [SEQ ID NO:45]
15
ON-3


NG-2
TTCCAGAACCACG [SEQ ID NO:46]
13
ON-3


NG-3
TCCAGAACCAC [SEQ ID NO:47]
11
ON-4


GP-5
GTTCCTGGCGCAG [SEQ ID NO:48]
13
ON-4


GP-6
TTCCTGGCGCAG [SEQ ID NO:49]
12
ON-4
















TABLE 5







Capture Sequence Probes for HSV-2













Location




Size
within


Probe
Sequence
(bp)
HSV-2





NF-1
GCCCGCGCCGCCAGCACTACTTTC
24
41610-



[SEQ ID NO:50]

41587


FG-1
AAACGTTGGGAGGTGTGTGCGTCATCC
35
48200-



TGGAGCTA [SEQ ID NO:51]

48234


LE-3
GACCAAAACCGAGTGAGGTTCTGTGT
26
48732-



[SEQ ID NO:52]

48757


NF-2
AAACGTTGGGAGGTGTGTGCGTCA
24
48200-



[SEQ ID NO:53]

48223


RA-3
TGCTCGTCACGAAGTCACTCATG
23
22756-



[SEQ ID NO:54]

22734


ON-2
CATTACTGCCCGCACCGGACC
21
23862-



[SEQ ID NO:55]

23842


LE-1
GCCGTGGTGTTCCTGAACACCAGG
24
27666-



[SEQ ID NO:56]

27643


LE-4
AGTCAGGGTTGCCCGACTTCGTCAC
25
22891-



[SEQ ID NO:57]

22867


NF-3
CAGGCGTCCTCGGTCTCGGGCGGGGC
26
32847-



[SEQ ID NO:58]

32822


NF-4
CCCACGTCACCGGGGGCCCC
20
26743-



[SEQ ID NO:59]

26724


LE-2
GCCGGTCGCGTGCGACGCCCAAGGC
25
33130-



[SEQ ID NO:60]

33106


SG-3
CCGACGCGTGGGTATCTAGGGGGTCG
26
90559-



[SEQ ID NO:61]

90534


SG-4
CGGGACGGCGAGCGGAAAGTCAACGT
26
86194-



[SEQ ID NO:62]

86169
















TABLE 6







Blocker Probes for HSV-2













Capture





Probe to


Probe

Size
which it


Name
Sequence
(bp)
hybridizes













HX-4
GGCGCGGGC [SEQ ID NO:63]
9
NF-1


HX-5
GAAAGTAGTGCTGGC [SEQ ID NO:64]
15
NF-1


GP-7
TGCTGGCGGCG [SEQ ID NO:65]
11
NF-1


AZ-3
ACACCTCCCAACG [SEQ ID NO:66]
13
FG-1


AZ-4
CTCCAGGATGACG [SEQ ID NO:67]
13
FG-1


GR-1
TCGGTTTTGGTC [SEQ ID NO:68]
12
LE-3


GR-2
ACACAGAACCTCA [SEQ ID NO:69]
13
LE-3


GP-8
CACACACCTCCCA [SEQ ID NO:70]
13
NF-2


BR-10
CGACCCCCTAGATA [SEQ ID NO:71]
14
SG-3


BR-11
CCACGCGTCGG [SEQ ID NO:72]
11
SG-3


HX-6
ACGTTGACTTTCCGC [SEQ ID NO:73]
15
SG-4


BR-15
CGCCGTCCCG [SEQ ID NO:74]
10
SG-4
















TABLE 7







Capture Sequence Probes for HPV












Size
HPV Type and


Probe
Sequence
(bp)
Sequence Location













ZL-1
GTACAGATGGTACCGGGGTTGTAGAAGTATCTG
33
HPV16



[SEQ ID NO:75]

5360-5392


ZL-4
CTGCAACAAGACATACATCGACCGGTCCACC
31
HPV16



[SEQ ID NO:76]

495-525


DP-1
GAAGTAGGTGAGGCTGCATGTGAAGTGGTAG
31
HPV16



[SEQ ID NO:77]

5285-5315


DP-4
CAGCTCTGTGCATAACTGTGGTAACTTTCTGGG
33
HPV16



[SEQ ID NO:78]

128-160


SH-1
GAGGTCTTCTCCAACATGCTATGCAACGTCCTG
33
HPV31



[SEQ ID NO:79]

505-537


SH-4
GTGTAGGTGCATGCTCTATAGGTACATCAGGCC
33
HPV31



[SEQ ID NO:80]

5387-5419


VS-1
CAATGCCGAGCTTAGTTCATGCAATTTCCGAGG
33
HPV31



[SEQ ID NO:81]

132-164


VS-4
GAAGTAGTAGTTGCAGACGCCCCTAAAGGTTGC
33
HPV31



[SEQ ID NO:82]

5175-5207


AH-1
GAACGCGATGGTACAGGCACTGCAGGGTCC
30
HPV18



[SEQ ID NO:83]

5308-5337


AH-2
GAACGCGATGGTACAGGCACTGCA
24
HPV18



[SEQ ID NO:84]

5314-5337


AL-1
ACGCCCACCCAATGGAATGTACCC
24
HPV18



[SEQ ID NO:85]

4451-4474


PA-4
TCTGCGTCGTTGGAGTCGTTCCTGTCGTGCTC
32
HPV18



[SEQ ID NO:86]

535-566


18-1AB
(TTATTATTA)CTACATACATTGCCGCCATGTTCG
36
HPV18



CCA [SEQ ID NO:87]

1369-1395


18-2AB
(TTATTATTA)TGTTGCCCTCTGTGCCCCCGTTGT
46
HPV18



CTATAGCCTCCGT [SEQ ID NO:88]

1406-1442


18-3AB
(TTATTATTA)GGAGCAGTGCCCAAAAGATTAAA
38
HPV18



GTTTGC [SEQ ID NO:89]

7524-7552


18-4AB
(TTATTATTA)CACGGTGCTGGAATACGGTGAGG
37
HPV18



GGGTG [SEQ ID NO:90]

3485-3512


18-5AB
(TTATTATTA)ACGCCCACCCAATGGAATGTACCC
33
HPV18



[SEQ ID NO:91]

4451-4474


18-6AB
(TTATTATTA)ATAGTATTGTGGTGTGTTTCTCAC
35
HPV18



AT [SEQ ID NO:92]

 81-106


18-7AB
(TTATTATTA)GTTGGAGTCGTTCCTGTCGTG
30
HPV18



[SEQ ID NO:93]

538-558


18-8AB
(TTATTATTA)CGGAATTTCATTTTGGGGCTCT
31
HPV18



[SEQ ID NO:94]

634-655


PE-1
GCTCGAAGGTCGTCTGCTGAGCTTTCTACTACT
33
HPV18



[SEQ ID NO:95]

811-843


PZ-2
GCGCCATCCTGTAATGCACTTTTCCACAAAGC
32
HPV45



[SEQ ID NO:96]

 77-108


PZ-5
TAGTGCTAGGTGTAGTGGACGCAGGAGGTGG
31
HPV45



[SEQ ID NO:97]

5295-5325


CS-1
GGTCACAACATGTATTACACTGCCCTCGGTAC
32
HPV45



[SEQ ID NO:98]

500-531


CS-4
CCTACGTCTGCGAAGTCTTTCTTGCCGTGCC
31
HPV45



[SEQ ID NO:99]

533-563


PF-1
CTGCATTGTCACTACTATCCCCACCACTACTTTG
34
HPV45



[SEQ ID NO:100]

1406-1439


PF-4
CCACAAGGCACATTCATACATACACGCACGCA
32
HPV45



[SEQ ID NO:101]

7243-7274


PA-1
GTTCTAAGGTCCTCTGCCGAGCTCTCTACTGTA
33
HPV45



[SEQ ID NO:102]

811-843


45-5AB
(TTATTATTA)TGCGGTTTTGGGGGTCGACGTGGA
36
HPV45



GGC [SEQ ID NO:103]

3444-3470


45-6AB
(TTATTATTA)AGACCTGCCCCCTAAGGGTACATA
36
HPV45



GCC [SEQ ID NO:104]

4443-4469


45-8AB
(TTATTATTA)CAGCATTGCAGCCTTTTTGTTACT
49
HPV45



TGCTTGTAATAGCTCC [SEQ ID NO:105]

1477-1516


45-9AB
(TTATTATTA)ATCCTGTAATGCACTTTTCCACAAA
34
HPV45



[SEQ ID NO:106]

 79-103


45-10AB
(TTATTATTA)GCCTGGTCACAACATGTATTAC
31
HPV45



[SEQ ID NO:107]

514-535


45-11AB
(TTATTATTA)CAGGATCTAATTCATTCTGAGGTT
33
HPV45



[SEQ ID NO:108]

633-656


ON-1
TGCGGTTTTGGGGGTCGACGTGGAGGC
27
HPV45



[SEQ ID NO:109]

3444-3470





*Sequences in parentheses are “tail” sequences not directed at HSV.













TABLE 8







Blocker Probes For HPV












Size
Capture Probe to


Probe
Sequence
(bp)
which it hybridizes













PV-FD-1
GCCTCCACGTCGAC [SEQ ID NO:110]
14
ON-1/45-5AB


PV-FD-2
CCCCAAAACCG [SEQ ID NO:111]
11
ON-1/45-5AB


PV-FD-3
GGTACATTCCATTGGG [SEQ ID NO:112]
16
18-5AB/AL-1


PV-FD-4
TGGGCGTTAATAATAA [SEQ ID NO:113]
16
18-5AB


AH-3
ACCATCGCGTTC [SEQ ID NO:114]
12
AH-2


AH-4
GGACCCTGCAGTGC [SEQ ID NO:115]
14
AH-1


AH-5
CTGTACCATCGCGTT 3′ [SEQ ID NO:116]
15
AH-1


AH-6
TGCAGTGCCTGT [SEQ ID NO:117]
12
AH-2


PZ-1
CCACCTCCTGCGT [SEQ ID NO:118]
13
PZ-5


PZ-3
ATTACAGGATGGCGC [SEQ ID NO:119]
15
PZ-2


PZ-4
GCTTTGTGGAAAAGTG [SEQ ID NO:120]
16
PZ-2


PZ-6
CCACTACACCTAGCACTA [SEQ ID NO:121]
18
PZ-5


ZL-2
CAGATACTTCTACAACC [SEQ ID NO:122]
17
ZL-1


ZL-3
CCGGTACCATCTGTAC [SEQ ID NO:123]
16
ZL-1


ZL-5
GGTGGACCGGTCG [SEQ ID NO:124]
13
ZL-4


ZL-6
ATGTATGTCTTGTTGCAG [SEQ ID NO:125]
18
ZL-4


DP-2
CTACCACTTCACATGC [SEQ ID NO:126]
16
DP-1


DP-3
AGCCTCACCTACTTC [SEQ ID NO:127]
15
DP-1


DP-5
CCCAGAAAGTTACCAC [SEQ ID NO:128]
16
DP-4


DP-6
AGTTATGCACAGAGCT [SEQ ID NO:129]
16
DP-4


SH-2
CAGGACGTTGCATAGC [SEQ ID NO:130]
16
SH-1


SH-3
ATGTTGGAGAAGACCTC [SEQ ID NO:131]
17
SH-1


SH-5
GGCCTGATGTACCTATA [SEQ ID NO:132]
17
SH-4


SH-6
GAGCATGCACCTACAC [SEQ ID NO:133]
16
SH-4


VS-2
CTCGGAAATTGCATG [SEQ ID NO:134]
15
VS-1


VS-3
AACTAAGCTCGGCATT [SEQ ID NO:135]
16
VS-1


VS-5
GCAACCTTTAGGGG [SEQ ID NO:136]
14
VS-4


VS-6
CGTCTGCAACTACTACTTC [SEQ ID NO:137]
19
VS-4


CS-2
GTACCGAGGGCAGT [SEQ ID NO:138]
14
CS-1


CS-3
GTAATACATGTTGTGACC [SEQ ID NO:139]
18
CS-1


CS-5
GGCACGGCAAGAAA [SEQ ID NO:140]
14
CS-4


CS-6
GACTTCGCAGACGTAGG [SEQ ID NO:141]
17
CS-4


PF-2
CAAAGTAGTGGTGGG [SEQ ID NO:142]
15
PF-1


PF-3
GATAGTAGTGACAATGCAG [SEQ ID NO:143]
19
PF-1


PF-5
TGCGTGCGTGTATGTA [SEQ ID NO:144]
16
PF-4


PF-6
TGAATGTGCCTTGTGG [SEQ ID NO:145]
16
PF-4


PE-2
AGTAGTAGAAAGCTCAGC [SEQ ID NO:146]
18
PE-1


PE-3
AGACGACCTTCGAGC [SEQ ID NO:147]
15
PE-1


PA-2
TACAGTAGAGAGCTCGG [SEQ ID NO:148]
17
PA-1


PA-3
CAGAGGACCTTAGAAC [SEQ ID NO:149]
16
PA-1


PA-5
GAGCACGACAGGAACG [SEQ ID NO:150]
16
PA-4


PA-6
ACTCCAACGACGCAGA [SEQ ID NO:151]
16
PA-4









EXAMPLE 3
Effect of the Extent of Biotin Labeling on Capture Efficiency
Tests were conducted to determine the optimal number of biotin labels per capture sequence probe for TSHC detection. The general TSHC method described in Example 1 was employed. The capture efficiency of capture sequence probe F15R labelled with one, two, or three biotins, measured by signal to noise ratio (S/N), were tested. The signal sequence probe employed was H19. As shown in Table 9, two biotins per capture sequence probe were sufficient for optimal capture efficiency. Greater than a 50% increase in S/N was observed using capture sequence probe with two biotin labels compared to the single biotin labeled capture sequence probe. The addition of a third biotin label to the capture sequence probe resulted in a decrease in S/N relative to the two-biotin labeled capture sequence probe.







TABLE 9







Effect of the Extent of Biotin Labeling on Capture Efficiency











# Biotins
HSV-1/well
RLU
CV
S/N














One
0
54
3%
1.0


One
4.5 × 10{circumflex over ( )}3
236
2%
4.4


One
4.5 × 10{circumflex over ( )}4
1861
3%
34.5


One
4.5 × 10{circumflex over ( )}5
15633
7%
289.5


Two
0
46
3%
1.0


Two
4.5 × 10{circumflex over ( )}3
296
10% 
6.4


Two
4.5 × 10{circumflex over ( )}4
2558
1%
55.6


Two
4.5 × 10{circumflex over ( )}5
23369
4%
508.0


Three
0
44
22% 
1.0


Three
4.5 × 10{circumflex over ( )}3
243
6%
5.5


Three
4.S × 10{circumflex over ( )}4
1820
2%
51.4


Three
4.5 × 10{circumflex over ( )}5
18581
8%
422.3









EXAMPLE 4
Effect of the Distance between the CSP and the SSP Target Sites on Capture Efficiency
The effect of the distance between capture sequence probe (CSP) and signal sequence probe (SSP) hybridization sites on a HSV-1 target nucleic acid on capture efficiency was evaluated. CSPs that hybridize to HSV-1 nucleic acid sequences which are located 0.2 kb, 3 kb, 18 kb, 36 kb and 46 kb from the site of SSP hybridization were tested. The general TSHC method described in Example 1 was employed. The capture efficiencies were 100%, 50%, 30%, 19% and 7%, respectively (Table 10). A steady decline in relative capture efficiencies was observed as the distance increased from 0.2 Kb to 46 Kb.







TABLE 10







Effect of Distance between Target Sites on Capture Efficiency














Distance Between
Relative Capture



CSP
SSP
Target Site
Efficiency







BRH19
H19
0.2 Kb
100% 



F15R
H19
  3 Kb
50%



F6R
RH5B
 18 Kb
30%



F15R
RH5B
 36 Kb
19%



F6R
H19
 46 Kb
 7% 










EXAMPLE 5
Effect of Fused Capture Sequence Probe on TSHC Detection of HSV-1
The binding capacity of streptavidin plates was determined to be approximately 2 pmoles of doubly-biotinylated CSPs per well. Since the CSPs are doubly biotin-labeled, a maximum of 8 CSPs (2 CSPs per SSP) is preferred in order not to exceed the binding capacity of the wells. Any increase in biotin-labeled capture sequence probe above the stated capacity resulted in a decrease in signal, the so-called “hook effect.” In order to avoid this “hook effect” and still permit the use of greater than four SSP-CSP combinations, the effect of synthesizing oligonucleotides that contained the sequences of two CSPs fused together (5′ and 3′ sites) was tested. The fused capture sequence probes may function independently to drive hybridization to the unique target sites. In another embodiment, the fused probes may bind to two target sites with the second hybridization favored, since it is essentially a uni-molecular reaction with zero order kinetics once the probe has hybridized to the first site. The hybridization may be determined by one or both mechanisms. Previous experiments showed that two CSPs, VH3 and NC-1, when used together, gave approximately twice the S/N as the individual CSPs. Unfused capture sequence probes VH-3 and NC-1 were used at 2.5 pmoles/ml each for a total concentration of 5 pmoles/ml, fused probe VH-4 (fusion of VH-3 and NC-1) was used at 2.5 pmole/ml. As shown in Table 11, the fused probe was as effective as the combination of the two unfused probes. Therefore, TSHC detection using fused capture sequence probes permits the number of nucleic acid sequences targeted by the signal sequence probe to be at least doubled without exceeding the plate biotin-binding capacity. The experiment also demonstrates the lack of cross-reactivity of HSV-2 at 107 genomes as shown by the S/N less than 2.0.







TABLE 11







Comparison of Fused v. Unfused Capture Sequence Probes


in TSHC Detection of HSV-1












SSP
CSP
Viral Particles/ml
RLU
CV
S/N















RH5B
VH-3, NC-1
0
94
14% 
1.0


RH5B
VH-3, NC-1
10{circumflex over ( )}4 HSV-1
164
5%
1.7


RH5B
VH-3, NC-1
10{circumflex over ( )}5 HSV-1
1003
4%
10.7


RH5B
VH-3, NC-1
10{circumflex over ( )}7 HSV-2
125
6%
1.3


RH5B
VH-4 (fused)
0
97
10% 
1.0


RH5B
VH-4 (fused)
10{circumflex over ( )}4 HSV-1
181
3%
1.9


RH5B
VH-4 (fused)
10{circumflex over ( )}5 HSV-1
1070
2%
11.0


RH5B
VH-4 (fused)
10{circumflex over ( )}7 HSV-2
140
5%
1.4









EXAMPLE 6
Capture Efficiency of Various CSPs and SSPs in TSHC Detection of HSV-1
The capture efficiency of capture sequence probes (CSPs) for each of the four HSV-1 specific signal sequence probes (SSPs), H19, RH5B, RH3 and RIO, in the detection of HSV-1 by TSHC were evaluated. The criteria used for designing the capture sequence probes were: 1) the CSP hybridization site is within 1 kb either 5′ or 3′ of the SSP hybridization site on the HSV-1 nucleic acid sequence, preferably within 0.5 kb; and 2) the CSPs contain sequences that are unique to HSV-1, with no stretches of sequence homology to HSV-2 greater than 10 bases. The CSPs were designed to target the 5′ and 3′ regions adjacent to the SSP hybridization site, preferably with a 5′ CSP and a 3′ CSP for each SSP. The Omiga software (Oxford Molecular Group, Campbell, Calif.) was instrumental in the identification of such sites. The melting temperature (Tm) of the CSPs was designed to be between 70° C. to 85° C., to conform to the 70° C. to 75° C. hybridization temperature used in Hybrid Capture II (HCII) assay for HSV (Digene). The general TSHC method described in Example 1 was employed. Eleven CSPs (which bind to 6 different sites) for H19, six CSPs (which bind to three unique sites) for RH5B, six CSPs (which bind to six unique sites) for RH3, and two CSPs for RIO were tested. As shown in Table 12, efficient capture sequence probes were found for signal sequence probes H19, RH5B and R10.







TABLE 12







CSPs and SSPs for TSHC Detection of HSV-1























Cap


SSP
CSP
Cap %
SSP
CSP
Cap %
SSP
CSP
%





R10
ON-3
100%
RH5B
TS-1
50%
H19
HZ-1
50%


R10
ON-3
 80%
RH5B
NC-1
75%
H19
HZ-2
20%





RH5B
VH-4
130% 
H19
ZD-1
40%





RH5B
TS-2
25%
H19
ZD-2
20%





RH5B
VH-3
50%
H19
BRH19
70%








H19
VH-2
70%








H19
F15R
25%









EXAMPLE 7
Capture Efficiency of Various CSPs and SSPs in TSHC Detection of HSV-2
The capture efficiency of capture sequence probes (CSPs) for each of the four HSV-2 specific signal sequence probes (SSPs), E4A, E4B, Ei8, and i8, in the detection of HSV-2 by TSHC were evaluated. HSV-2 specific capture sequence probes (CSPs) were designed based on the same criteria as the HSV-1 CSPs except for the requirement that they be HSV-2 specific. Four CSPs for E4A, three CSPs for E4B, and two CSPs each for Ei8 and i8 were tested. The general TSHC method described in Example 1 was employed. As shown in Table 13, efficient capture sequence probes were found for i8 and Ei8.







TABLE 13







CSPs and SSPs for TSHC Detection of HSV-2














SSP
CSP
Cap%
SSP
CSP
Cap%







18
NF-1
100%
Ei8
NF-2
50%






Ei8
LE-3
45%










EXAMPLE 8
Effect of Blocker Probes on HSV-1 and HSV-2 Detection
In an attempt to reduce cross-reactivity of TSHC while allowing the capture step to take place at room temperature, methods using blocker probes were developed. Blocker probes comprise sequences that are complementary to the capture sequence probes (CSPs) used for detection. These experiments were designed to prevent non-specific hybridization of the CSPs to non-targeted nucleic acids present in the sample under the lower stringency conditions, a situation often encountered during the room temperature capture step.
In one method, blocker probes that are complementary to the full length or nearly the full length of the capture sequences probe were used. The blocker probes were added to the reaction mixture in 10-fold excess relative to the CSP after hybridization of the CSP and the SSP to the target DNA molecule has occurred. Since the blocker probes have similar melting temperature as the CSPs, the CSPs were hybridized to the target nucleic acids first to prevent hybridization of the blocker probes to the CSPs before the hybridization of the CSPs to the target nucleic acids occurred. As shown in Table 14, the addition of the blocker probes resulted in a dramatic reduction in cross-reactivity while these probes had no effect on the sensitivity of HSV-1 detection. The S/N for the detection of cross-reactive HSV-2 (107 viral particles/ml) decreased from 5.0 to 0.8 when the blocker probes were used.
In another method, blocker probes that are complementary to only a portion of the CSPs and are shorter than the CSPs were used. The blocker probes were designed to have melting temperatures above room temperature but at least 10° C. below the hybridization temperature of CSPs to the target nucleic acids. Since these blocker probes hybridize to the CSPs at temperature below the CSP hybridization temperature to the target nucleic acids, the blocker probes may be added to the reaction at the same time as the CSP and SSP without effecting the hybridization efficiency of the CSPs to the target nucleic acid. These shorter blocker probes function during the room temperature capture step by hybridizing to the CSPs at the lower temperatures that are encountered during the room temperature capture step. As shown in Table 15, the addition of either single or paired shorter blocker probes in 100-fold excess relative to the CSPs resulted in a dramatic reduction in cross-reactivity but had no effect on sensitivity of HSV-1 detection. The S/N for detecting cross-reactive HSV-2 (107 viral particles/ml) without the blocker probes was 10.6, but was reduced to less than or equal to 1.5 with the addition of the blocker probes.
Therefore, both methods utilizing blocker probes provide a substantial reduction in cross-reactivity. The second method utilizing blocker probes with lower melting temperature may be preferred because the addition of blocker probes at the same time as the capture sequence probe eliminates the need for an extra step for the detection method.







TABLE 14







Effect of Blocker Probes Added Post Capture probe


hybridization on TSHC















100×






SSP
CSP
Blocker Probe
Viral Particles/ml
RLU
CV
S/N
















H19
HZ-1
None
0
66
7%
1.0


H19
HZ-1
None
10{circumflex over ( )}5 HSV-1
246
5%
3.7


H19
HZ-1
None
10{circumflex over ( )}6 HSV-1
1998
2%
30.3


H19
HZ-1
None
10{circumflex over ( )}7 HSV-2
327
2%
5.0


H19
HZ-1
ZD-3
0
60
3%
1.0


H19
HZ-1
ZD-3
10{circumflex over ( )}5 HSV-1
267
4%
4.5


H19
HZ-1
ZD-3
10{circumflex over ( )}6 HSV-1
2316
6%
38.6


H19
HZ-1
ZD-3
10{circumflex over ( )}7 HSV-2
49
2%
0.8
















TABLE 15







Effect of Blocker Probes Added Simultaneously with the


Capture Probes on TSHC Detection of HSV-1















10×






SSP
CSP
Blocker Probe
Viral Particles/ml
RLU
CV
S/N
















H19
HZ-1
none
0
38
15%
1.0


H19
HZ-1
none
10{circumflex over ( )}4 HSV-1
71
 2%
1.9


H19
HZ-1
none
10{circumflex over ( )}5 HSV-1
389
12%
10.2


H19
HZ-1
none
10{circumflex over ( )}7 HSV-2
401
18%
10.6


H19
HZ-1
NG-4
0
39
 8%
1.0


H19
HZ-1
NG-4
10{circumflex over ( )}4 HSV-l
82
 5%
2.1


H19
HZ-1
NG-4
10{circumflex over ( )}5 HSV-1
411
18%
10.5


H19
HZ-1
NG-4
10{circumflex over ( )}7 HSV-2
57
15%
1.5


H19
HZ-1
EA-1, EA-2
0
37
 0%
1.0


H19
HZ-1
EA-1, EA-2
10{circumflex over ( )}4 HSV-1
75
 8%
2.0


H19
HZ-1
EA-1, EA-2
10{circumflex over ( )}5 HSV.1
419
 8%
11.3


H19
HZ-1
EA-1, EA-2
10{circumflex over ( )}7 HSV-2
49
 5%
1.3


H19
HZ-1
NG-7, NG-8
0
42
10%
1.0


H19
HZ-1
NG-7, NG-8
10{circumflex over ( )}4 HSV-1
76
 3%
1.8


H19
HZ-1
NG-7, NG-8
10{circumflex over ( )}5 HSV-1
471
 5%
11.2


H19
HZ-1
NG-7, NG-8
10{circumflex over ( )}7 HSV-2
47
 9%
1.1









EXAMPLE 9
TSHC Detection Reduces Vector Background
The TSHC assay eliminates the vector contamination problem often associated with the Hybrid Capture II (HC II) detection assay (Digene). As the RNA signal sequence probes used in HC II are generated from linearized vector templates, any remaining unlinearized plasmid DNA results in the production of additional RNA probe sequences specific for vector sequences. In the HC II assay, the RNA/DNA hybrids that form as a result of these read-through transcripts are captured on the antibody coated plates and generate signal. In contrast, in the TSHC method, only those RNA/DNA hybrids that also hybridize to the capture sequence probes are detected. Accordingly, any detection of vector-related sequences is eliminated. Plasmids SK+, pBR322, DgZ and 1066 which were known to be detectable in HSV HC II test (Digene) were tested in the TSHC assay using two RNA signal sequence probes (H19 and RH5b) and two capture sequence probes (VH-2 and VH-4). Identical set of RNA probes were then used in HC II method and the TSHC method for the detection of HSV-1. The general TSHC method described in Example 1 was employed. As shown in Table 16, while signal to noise ratio in standard HC II ranged from 14 to 48, the signal to noise ratio for the TSHC method was less than 2 for all plasmids tested.







TABLE 16







Vector Background in TSHC v. HCII Detection













Method
SSP
CSP
Targets/ml
RLU
CV
S/N
















TSHC
H19 + RH5B
VH-2 + VH-4
0
94
6%
1.0


TSHC
H19 + RH5B
VH-2 + VH-4
4 ng pBS
137
7%
1.5





SK+


TSHC
H19 + RH5B
VH-2 + VH-4
2 ng
99
6%
1.1





pBR322


TSHC
H19 + RH5B
VH-2 + VH-4
4 ng DgX
135
7%
1.4


TSHC
H19 + RH5B
VH-2 + VH-4
4 ng 1066
107
7%
1.1


HC II
H19 + RH5B
None
0
94
9%
1.0


HC II
H19 + RH5B
None
4 ng pBS
4498
3%
48.1





SK+


HC II
H19 + RH5B
None
2 ng
1281
8%
13.7





pBR322


HC II
H19 + RH5B
None
4 ng DgX
2003
5%
21.4


HC II
H19 + RH5B
None
4 ng 1066
1536
2%
16.4









EXAMPLE 10
Sensitivity and Specificity of detecting HSV-1 and HSV-2 by TSHC
The sensitivity and typing discrimination for the TSHC detection of HSV-1 and HSV-2 were assessed using the TSHC described in Example 1. In the HSV-1 TSHC assay, signal sequence probes H19 and RH5B, capture sequence probes HZ-1, VH-2 and VH-4, and blocker probes NG-7, NG-8, GP-3, GP-4, and GP-1 were used. In the HSV-2 TSHC assay, signal sequence probes I8 and Ei8, capture sequence probes NF-1 and NF-2, and blocker probes HX-4, HX-5 and GP-8 were used. HSV-1 and HSV-2 viral particles were diluted to various concentrations using the Negative Control Solution. As shown in FIGS. 4 and 5, while 104 copies of the either HSV-1 or HSV-2 (450 copies/well) were detected in the respective assays, there was virtually no detection of the cross-reactive type HSV at concentrations up to and including 108 copies/ml (4,500,000 copies/well). Thus, the HSV-1 and HSV-2 TSHC assays can distinguish the two HSV types at a greater than 10,000-fold range of discrimination while maintaining excellent sensitivity (450 VP/well).
The HSV-1 TSHC assay shows a linear range of detection ranging from at least 2×103 to 5×103 VP/ml (Table 17). The specificity of the assay is excellent as no cross-reactivity was detected (S/N is less than or equal to 2) in samples containing HSV-2 at a concentration as high as 2×107 to 5×107 viral particles/ml. Similarly, the HSV-2 TSHC assay also shows excellent specificity, wherein no cross-reactivity was detected in samples containing HSV-1 at a concentration as high as 5×107 viral particles/ml (Table 18). Similar results were obtained from TSHC detection of HSV-2 using a dilution series of HSV-2 and HSV-1 viruses (Table 19).







TABLE 17







Analytical Sensitivity and Specificity of the HSV1 TSHC Assay









Targets
RLU
S/N












Negative Control
47
1.0


HSV2 @ 5 × 10{circumflex over ( )}7 VP/ml
57
1.2


HSV2 @ 2 × 10{circumflex over ( )}7 VP/ml
43
0.9


HSV1 @ 5 × 10{circumflex over ( )}3 VP/ml
201
4.3


HSV1 @ 2 × 10{circumflex over ( )}3 VP/ml
107
2.3
















TABLE 18







Analytical Sensitivity and Specificity of the HSV2 TSHC Assay









Targets
RLU
S/N












Negative Control
40
1.0


HSV1 @ 5 × 10{circumflex over ( )}7 VP/ml
78
2.0


HSV1 @ 2 × 10{circumflex over ( )}7 VP/ml
55
1.4


HSV2 @ 5 × 10{circumflex over ( )}3 VP/ml
218
5.5


HSV2 @ 2 × 10{circumflex over ( )}3 VP/ml
106
2.7
















TABLE 19







Detection with HSV-2 Probes using HSV-1


and HSV-2 of Different Dilution









Targets
RLU
S/N












Negative Control
43
1.0


HSV1 @ 5 × 10{circumflex over ( )}7 VP/ml
112
2.6


HSV1 @ 2 × 10{circumflex over ( )}7 VP/ml
57
1.3


HSV1 @ 1 × 10{circumflex over ( )}7 VP/ml
38
0.9


HSV1 @ 1 × 10{circumflex over ( )}6 VP/ml
38
0.9


HSV1 @ 1 × 10{circumflex over ( )}5 VP/ml
33
0.8


HSV1 @ 1 × 10{circumflex over ( )}4 VP/ml
52
1.2


HSV1 @ 1 × 10{circumflex over ( )}3 VP/ml
43
1.0


HSV1 @ 1 × 10{circumflex over ( )}2 VP/ml
39
0.9


HSV2 @ 1 × 10{circumflex over ( )}7 VP/ml
257173
5980.8


HSV2 @ 1 × 10{circumflex over ( )}6 VP/ml
28544
663.8


HSV2 @ 1 × 10{circumflex over ( )}5 VP/ml
3200
74.4


HSV2 @ 1 × 10{circumflex over ( )}4 VP/ml
266
6.2


HSV2 @ 5 × 10{circumflex over ( )}3 VP/ml
181
4.2


HSV2 @ 1 × 10{circumflex over ( )}3 VP/ml
62
1.4


HSV2 @ 1 × 10{circumflex over ( )}2 VP/ml
44
1.0









EXAMPLE 11
Clinical Specimen Testing
A 64-member clinical specimen panel was tested for HSV-1 and HSV-2 using both TSHC and HCII methods. The panel included 15 samples containing known quantities of HSV-1 or HSV-2, and 49 samples known to be negative for HSV-1 and HSV-2 by PCR testing. Accordingly, the 15 positive samples were “Expected” to test positive in both the HCII and TSHC assays, and the 49 negative samples were “Expected” to test negative in both the HCII and TSHC tests.
The general TSHC method described in Example 1 was employed. The results using the HCII method and the TSHC method are shown in Tables 20 and 21, respectively. Of the 49 samples “Expected” to yield negative result, 5 samples tested positive and 44 samples tested positive using the HCII method. In comparison, all 49 samples tested negative using the TSHC method. Therefore, the TSHC method is superior in specificity to the HCII method in the detection of HSV-1 and HSV-2.







TABLE 20







Observed vs. Expected Results for HCII Detection of HSV1 and HSV2










Expected Result










HCII Result
Positive
Negative












Positive
15
5


Negative
0
44


Total
15
49
















TABLE 21







Observed vs. Expected Results for TSHC Detection of HSV1 and HSV2










Expected Result










TSHC Result
Positive
Negative












Positive
14
0


Negative
1
49


Total
15
49









EXAMPLE 12
Effect of Combining Probes in TSHC Detection of HSV
The effect of combining HSV-1 specific signal sequence probe and capture sequence probe sets on HSV-1 detection was assessed. TSHC detection of HSV-1 and HSV-2 cross-reactivity was performed separately with two different sets of RNA signal sequence probe/biotinylated capture sequence probe combinations (Set # 1: H19 plus HZ-1; and Set #2: RH5b plus the TS-1 and TS-2). TSHC was also performed with both RNA signal sequence probe/biotinylated capture sequence probe sets combined to assess the effect of combining the two probe sets on sensitivity and cross-reactivity. The general TSHC method described in Example 1 was employed. The results shown in Table 22 clearly demonstrate an additive effect of combining the two probe sets for HSV-1 detection with no apparent increase in HSV-2 cross-reactivity.







TABLE 22







Sensitivity is Improved by Combining HSV-1 Specific CSPs and SSPs












Capture Sequence
Signal






Probes
Sequence Probes
VP/ml
RLU
CV
S/N















HZ-1
H19
0
60
3%
1.0


HZ-1
H19
10{circumflex over ( )}5 HSV-1
267
4%
4.5


HZ-1
H19
10{circumflex over ( )}6 HSV-1
2316
6%
38.9


HZ-1
H19
10{circumflex over ( )}7 HSV2
49
2%
0.8


TS-1, TS-2
RH5B
0
78
6%
1.0


TS-1, TS-2
RH5B
10{circumflex over ( )}5 HSV-1
291
6%
3.8


TS-1, TS-2
RH5B
10{circumflex over ( )}6 HSV-1
2368
11% 
30.6


TS-1, TS-2
RH5B
10{circumflex over ( )}7 HSV2
75
11% 
1.0


HZ-1, TS-1, TS-2
H19, RH5B
0
70
12% 
1.0


HZ-1, TS-1, TS-2
H19, RH5B
10{circumflex over ( )}5 HSV-1
457
10% 
6.5


HZ-1, TS-1, TS-2
H19, RH5B
10{circumflex over ( )}6 HSV-1
4263
1%
60.9


HZ-1, TS-1, TS-2
H19, RH5B
10{circumflex over ( )}7 HSV2
67
6%
1.0









EXAMPLE 13
TSHC Detection of HPV18 and HPV45
The relative sensitivity and specificity of TSHC and HCII detection of Human Papillomavirus 18 (HPV18) and Human Papillomavirus 45 (HPV45) was compared. Previous studies have established HPV45 as the most cross-reactive HPV type to HPV18, and conversely, HPV18 as the most cross-reactive HPV type to HPV45. In this study, the ability of the two methods to detect HPV18 and HPV45 was assessed using HPV18 and HPV45 plasmid DNA.
Capture sequence probes (CSPs) for each of the four Human Papillomavirus types: HPV16, HPV18, HPV31, and HPV45, were designed. The criteria used for designing the capture sequence probes were: 1) the CSP hybridization istes do not overlap with the SSP sites; 2) the CSPs contain sequences unique to one HPV type with no stretches of sequence homology to other HPV types greater than 12 bases; and 3) the CSPs are of sufficient length so as to be capable of hybridizing efficiently at 70° C.
The blocker probes for each CSP were designed such that they could be added simultaneously with the CSP during hybridization to the target nucleic acid. The blocker probes have a melting temperature of at least 37° C. but no higher than 60° C., as calculated by the Oligo 5.0 program (National Biosciences, Inc., Plymouth, Minn.). Two blocker probes were used for each capture oligonucleotide to maximize the blocker effect during the room temperature plate capture step. It was also desired that the blocker probes for each CSP have similar melting temperatures.
CSPs for each of the HPV types were tested for relative capture efficiency and cross-reactivity to other HPV types. CSPs that provided the best combination of sensitivity and low cross-reactivity were used for the detection of HPV using TSHC.
In TSHC and HCII detection of HPV18, HPV18 DNA was used at a concentration of 10 pg/ml. HPV45, used for cross-reactivity testing, was used at 4 ng/ml. The general TSHC method described in Example 1 was employed. As shown in Table 23, a signal to noise ratio of 16.9 was obtained for TSHC detection of HPV18 compared to a ratio of 7.6 obtained for HCII detection of HPV18. On the other hand, cross-reactivity with HPV45 was significantly reduced using the TSHC method (S/N of 1.3 for TSHC compared to S/N of 393.3 for HCII). The results clearly show that compared to the HCII method, the TSHC method for the detection of HPV18 was superior in both sensitivity and specificity. Results obtained in experiments comparing TSHC and HCII detection of HPV45 demonstrate that the TSHC method for the detection of HPV45 is superior in both sensitivity and specificity (Table 24).







TABLE 23







TSHC Detection of HPV 18











Method
Target
SSP
CSP
S/N














TSHC
0
18L1
18-7L
1.0



HPV18 (10 pg/ml)
18L1
18-7L
16.9



HPV45 (4 ng/ml)
18L1
18-7L
1.3


HC II
0
18L1
none
1.0



HPV18 (10 pg/ml)
18L1
none
7.6



HPV45 (4 ng/ml)
18L1
none
393.3
















TABLE 24







TSHC Detection of HPV 45











Method
Target
SSP
CSP
S/N














TSHC
0
45L1
ON-1
1.0



HPV45 (10 pg/ml)
45L1
ON-1
8.4



HPV18 (4 ng/ml)
45L1
ON-1
1.6


HC II
0
45L1
none
1.0



HPV45 (10 pg/ml)
45L1
none
8.2



HPV18 (4 ng/ml)
45L1
none
494.0









EXAMPLE 14
Target-Specific Hybrid Capture-Plus Assay Protocol
Hepatitis B Virus (HBV) was used as the model system for the development of the target-specific hybrid capture-plus (TSHC-plus) assay for the detection of target nucleic acids.
The hybridization in the TSHC-plus method (FIGS. 6A-6D) may be performed in a single step. In the one-step method, CSPs, SSPs containing pre-hybridized DNA-RNA duplex, bridge probes (FIGS. 6B-6D), and blocker probes are added simultaneously to the target nucleic acids. If hybridization is performed in two steps, CSPs, SSPs without pre-hybridized DNA-RNA duplex, bridge probes and blocker probes are first hybridized to the target nucleic acid. Oligonucleotide probes complementary to the single stranded nucleic acid sequence in the SSP are then added to the reaction to form the DNA-RNA duplexes. The hybrids are then detected using anti-RNA/DNA antibody as described in Example 1.
Experiments were carried out to detect HBV using TSHC-plus (Examples 15-18). The method shown in FIG. 6A was used. Human hepatitis B virus (HBV adw2) plasmid DNA of known concentration (Digene Corp) was diluted using HBV negative Sample Diluent (Digene). Various dilutions were made and aliquoted into individual tubes. The negative Sample Diluent was used as a negative control. A half volume of the Denaturation Reagent 5100-0431 (Digene) was added to the test samples. Test samples were incubated at 65° C. for 45 minutes to denature the nucleic acids in the samples.
Following denaturation of the HBV sample, a hybridization solution containing capture sequence probes (CSPs), blocker probes, signal sequence probe comprising a M13 DNA/M13 RNA duplex and a single-stranded DNA sequence capable of hybridizing to HBV sequences was added to the samples, and incubated at 65° C. for 1-2 hours. Alternatively, the denatured samples were incubated for 1 hour with a hybridization solution containing capture sequence probes (CSPs), blocker probes and M13 DNA plasmid containing HBV complementary sequences for 1 hour. Following the incubation, M13 RNA was added to the reaction and the incubation was continued for an additional hour at 65° C.
Tubes containing reaction mixtures were cooled at room temperature for 5 minutes and aliquots were taken from each tube and transferred to individual wells of a 96-well streptavidin plate (Digene). The plates were shaken at 1100 rpms for 1 hour at room temperature. The solution was then decanted and the plates were washed four times with SNM wash buffer (Digene). The alkaline-phosphatase anti-RNA/DNA antibody DR-1 (Digene) was added to each well and incubated for 30 minutes at room temperature. The DR-1 (Digene) was then decanted and the plates were washed four times with SNM wash buffer (Digene). Following removal of the residual wash buffer, luminescent substrate (CDP-Star, Tropix Inc.) was added to each well and incubated for 15 minutes at room temperature. Individual wells were read on a plate luminometer to obtain relative light unit (RLU) signals.
EXAMPLE 15
The following tables describe the various probes tested in the experiments described in Examples 16-18.







TABLE 25







Capture Sequence Probes for HBV













Size
Location within



Probe
Sequence
(bp)
HBV
Strand





HBV C1
GCTGGATGTGTCTGCGGCGTTTTATCAT
28
374-401
Sense



(SEQ ID NO: 152)


HBV C2
ACTGTTCAAGCCTCCAAGCTGCGCCTT
27
1861-1877
Sense



(SEQ ID NO: 153)


HBV C3
ATGATAAAACGCCGCAGACACATCCAGCG
32
370-401
Anti-



ATA (SEQ ID NO: 154)


sense
















TABLE 26







HBV/M13 Clones from which SSPs are Prepared














Insert Size
Location


Clone name
Vector
Cloning site
(bp)
within HBV





SA1
M13 mp 18
Eco RI, Hind III
35
194-228


SA2
M13 mp 18
Eco RI, Hind III
34
249-282


SA1a
M13 mp 19
Eco RI, Hind III
35
194-228


SA2a
M13 mp 19
Eco RI, Hind III
34
249-282


SA4
M13 mp 19
Eco RI, Hind III
87
1521-1607
















TABLE 27







HBV Blocker probes













CSP




Size
to which it


Probe
Sequence
(bp)
hybridizes





B1
ATGATAAAACGCCG (SEQ ID NO: 155)
14
HBV C1


B2
CAGACACATCCAGC (SEQ ID NO: 156)
14
HBV C1


B3
AAGGCACAGCTTG (SEQ ID NO: 157)
13
HBV C2


B4
GAGGCTTGAACAGT (SEQ ID NO: 158)
14
HBV C2


B5
TATCGCTGGATGTGTC (SEQ ID NO: 159)
16
HBV C3


B6
TCGGCGTTTTATCATG (SEQ ID NO: 160)
16
HBV C3









EXAMPLE 16
Effect of Blocker Probes on TSHC-Plus Detection of HBV
During room temperature capture step, excess SSP (M13 RNA/HBV-M13 DNA duplex) non-specifically hybridizing to the CSP are immobilized onto the plate which results in high background signals. In an attempt to reduce background signal, blocker probes were employed in TSHC-Plus detection of HBV. The blocker probes were designed to be much shorter than the CSPs so that they are only capable of hybridizing to the capture probes at temperatures well below the hybridization temperatures used in the assay.
Blocker probe sets consisting of two separate oligonucleotides that are complementary to the CSPs were used. The blocker probes were added to the hybridization mixture in 10-fold excess relative to the CSPs. Since the blocker probes are much shorter than the CSPs, they do not hybridize with CSPs at the target hybridization temperature and therefore do not interfere with the hybridization of the CSPs to the target nucleic acids. Following the hybridization of CSP and target nucleic acids, the samples were subjected to a room temperature capture step during which the blocker probes hybridize with excess CSPs, thus preventing them from hybridizing to the SSPs. As shown in Table 28, the use of the blocker probes in the hybridization reaction greatly reduced the background signals of the assay.







TABLE 28







Effect of Blocker Probes on HBV Detection









Capture Probe
Blocker probe
Background Signal (RLU)












HBV C1
no
17892


HBV C1
B1, B2
424


HBV C2
no
9244


HBV C2
B3, B4
398









EXAMPLE 17
Effect of the Length of SSP on TSHC-Plus Detection of HBV
The effect of the length of the DNA sequence inserted into the M13 vector for generating the SSP on TSCH-Plus detection of HBV was studied. A positive control containing 20 pg/ml of HBV plasmid DNA was used. As shown in Table 29, the use of a longer HBV complementary sequence in the SSP (87 base pairs) resulted in a substantial increase in signal of detection. The effect is unlikely due to sub-optimal hybridization temperature condition since the Tm of the shorter probes is 15 degree above the hybridization temperature. As the M13 RNA-DNA duplex formed in the SSP may act to partially block the complementary DNA sequence in the probe from hybridizing to the HBV sequences in the target nucleic acids, longer complementary sequences in the SSP may overcome this block.







TABLE 29







Effect of the Length of the Complementary sequence in


the SSP on TSHC-Plus Detection of HBV












Size of the HBV






Target DNA
Tm of the HBV



Sequence in SSP
Target DNA
Hybridization
Signal


SSP
(bp)
Sequence in SSP
temperature
(RLU)





SA1
35
83° C.
65° C.
1741


SA2
34
80° C.
65° C.
1857


SA4
87
108° C. 
65° C.
7978









EXAMPLE 18
TSHC-Plus and HC II Detection of HBV
The relative sensitivity of TSHC-Plus and HC II (Hybrid Capture II, Digene) detection of HBV was compared. HBV positive standards of three different concentrations were tested in the experiments. As shown in Table 30, the signals obtained using the TSHC-Plus detection method were approximately two-fold higher than those obtained using the HC II detection method.







TABLE 30







TSHC-Plus and HC II Detection of HBV*









Target HBV Concentration











Method
Control
10 pg/ml
20 pg/ml
100 pg/ml














HC II
48
2355
4225
21438


TSHC Plus
285
4856
7978
37689





*Signal measured as relative light unit (RLU)






The above description of various preferred embodiments has been presented for purposes of illustration and description. It is not intended to be exhaustive or limiting to the precise forms disclosed. Obvious modifications or variations are possible in light of the above teachings. The embodiments discussed were chosen and described to provide illustrations and its practical application to thereby enable one of ordinary skill in the art to utilize the various embodiments and with various modifications as are suited to the particular use contemplated. All such modifications and variations are within the system as determined by the appended claims when interpreted in accordance with the breadth to which they are fairly, legally and equitably entitled.

## Claims

1. A method of detecting a target nucleic acid comprising:
a) hybridizing a single-stranded target nucleic acid to a capture sequence probe and a signal sequence probe to form a hybrid complex comprising double-stranded hybrids between said capture sequence probe and a portion of the target nucleic acid, and between said signal sequence probe and a portion of the target nucleic acid, wherein the capture sequence probe and the signal sequence probe hybridize to non-overlapping regions within the target nucleic acid and do not hybridize to each other, wherein the signal sequence probe is unlabeled; and
b) adding a blocker probe to the hybridization reaction, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes;
c) capturing the capture sequence probe:target portion of said hybrid complex to form a bound hybrid complex; and
d) detecting the bound double-stranded hybrid complex, thereby detecting the target nucleic acid.

2. A method of detecting a target nucleic acid comprising:
a) hybridizing a single-stranded target nucleic acid to an immobilized capture sequence probe and a signal sequence probe to form a hybrid complex comprising double-stranded hybrids between said immobilized capture sequence probe and a portion of the target nucleic acid, and between said signal sequence probe and a portion of the target nucleic acid, wherein the capture sequence probe and the signal sequence probe hybridize to non-overlapping regions within the target nucleic acid and do not hybridize to each other;
b) adding a blocker probe to the hybridization reaction, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes; and
c) detecting the immobilized double-stranded hybrid complex, thereby detecting the target nucleic acid.

3. The method of claim 1 or 2, wherein the capture sequence probe is modified with at least one ligand.

4. The method of claim 2, wherein the signal sequence probe is unlabeled.

5. The method of claim 3, wherein the ligand is biotin.

6. The method of claim 5, wherein the capture sequence probe is linear having a 5′ and 3′ end, wherein both the 5′ and the 3′ ends are biotinylated.

7. The method of claim 1 or 2, wherein the capture sequence probe and the signal sequence probe hybridize to regions of the target nucleic acid, wherein the regions are less than 3 kilobases apart.

8. The method of claim 1 or 2, wherein the capture sequence probe and the signal sequence probe hybridize to regions of the target nucleic acid, wherein the regions are less than 500 bases apart.

9. The method of claim 1 or 2, wherein the capture sequence probe is a fusion of two or more sequences complementary to different regions of the target nucleic acid or to different target molecules.

10. The method of claim 1 or 2, wherein the double-stranded hybrid formed is a DNA-RNA hybrid.

11. The method of claim 1 or 2, further comprising the step of forming single-stranded DNA prior to the hybridization step.

12. The method of claim 1 or 2, wherein hybridization of the capture sequence probe and the signal sequence probe to the target nucleic acid are performed sequentially.

13. The method of claim 1 or 2, wherein step a) and step b) are performed simultaneously.

14. The method of claim 1 or 2, wherein the blocker probe has lower melting temperature than that of the capture sequence probe.

15. The method of claim 1, wherein the hybrid is captured onto a solid phase.

16. The method of claim 15, wherein the solid phase is coated with streptavidin.

17. The method of claim 15, wherein the solid phase is a microplate.

18. The method of claim 1 or 2, wherein step c) is carried out at room temperature.

19. The method of claim 1 or 2, wherein the bound double-stranded hybrid is detected using an antibody which recognizes a hybrid.

20. The method of claim 19, wherein the hybrid is a DNA-RNA-hybrid.

21. The method of claim 20, wherein the antibody which recognizes a DNA-RNA hybrid is labeled with alkaline-phosphatase.

22. The method of claim 1, wherein the hybrid formed in step a) is captured onto a solid phase.

23. The method of claim 22, wherein the solid phase is coated with streptavidin.

24. The method of claim 22, wherein the solid phase is a microplate.

25. The method of claim 20, wherein the blocker probe is added to the hybridization reaction following the hybridization of the capture sequence probe to the target nucleic acid.

26. The method of claim 20, wherein the blocker probe has lower melting temperature than that of the capture sequence probe.

27. The method according to claim 1, wherein the signal sequence probe comprises a DNA-RNA duplex and a single-stranded nucleic acid sequence which hybridizes to the target nucleic acid.

28. The method according to claim 27, wherein the DNA-RNA duplex is a M13 DNA-M13 RNA duplex.

29. A method of detecting a target nucleic acid comprising:
a) hybridizing a single-stranded target nucleic acid to a capture sequence probe, a bridge probe and a signal sequence probe to form a hybrid complex comprising double-stranded hybrids between said capture sequence probe and a portion of the target nucleic acid, and between said bridge probe and a portion of the target nucleic acid, wherein the capture sequence probe and the bridge probe each hybridize to non-overlapping regions within the target nucleic acid and do not hybridize to each other, and the signal sequence probe hybridizes to the bridge probe and does not hybridize to the target nucleic acid and the capture sequence probe;
b) adding a blocker probe to the hybridization reaction, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes;
c) capturing the hybrid complex to form a bound double-stranded hybrid complex; and
d) detecting the bound double-stranded hybrid complex, thereby detecting the target nucleic acid.

30. The method according to claim 29, wherein the signal sequence probe comprises a DNA-RNA duplex and a single stranded nucleic acid which hybridizes to the bridge probe.

31. The method according to claim 30, wherein the DNA-RNA duplex is a M13 DNA-M13 RNA duplex.

32. The method according to claim 30, wherein the DNA-RNA duplex is a hybrid formed between repeat sequences within the signal sequence probe and a nucleic acid molecule having complementary sequences to the repeat sequences.

33. The method according to claim 29, wherein the bridge probe further comprises a poly(A) tail.

34. The method according to claim 33, wherein the signal sequence probe comprises a single stranded poly(dT) DNA sequence which hybridizes-to the poly(A) tail of the bridge probe, and a DNA-RNA duplex formed between the poly(dT) sequences in the signal sequence probe and a nucleic acid molecule having poly(A) sequences.