WO2007084567A2 - Detection and discrimination of hepatitis c virus, human immunodeficiency virus type-1 and hepatitis b virus - Google Patents

Abstract

The present disclosure provides isolated nucleic acid primers and probes that can be used to detect hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus type 1 (HIV-I), or combinations thereof (for example simultaneously), in a biological sample. In particular examples, the disclosed methods can discriminate 3-10 genomic equivalents (gEq) of HBV, 10-20 gEq of HCV, and 20 gEq of HIV-I. Also provided are arrays that include the viral-specific probes, as well as kits that include the isolated nucleic acid primers, probes, arrays, or combinations thereof. The method and arrays can be used for example, for diagnosis of these viruses in clinics and in high-risk groups, as discriminatory confirmation assay in screening of blood donors, and in research laboratories.

Description

DETECTION AND DISCRIMINATION OF HEPATITIS C VIRUS, HUMAN IMMUNODEFICIENCY VIRUS TYPE-I AND HEPATITIS B VIRUS

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 60/759,214 filed January 17, 2006, herein incorporated by reference.

FIELD

This application relates to methods of detecting hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus type 1 (HIV-I), as well as isolated nucleic acid molecules, arrays, and kits that can be used to detect these viruses.

BACKGROUND

Hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type-1 (HIV-I) are prevalent blood-borne viruses of worldwide public health concern. Approximately 1-1.25 million HBV chronic carriers live in the U.S., and about 2 billion individuals have been infected with this virus worldwide. There are about 3.9 million individuals infected with HCV in the U.S. and 170 million (3.5 % of world population) worldwide (Alter et aL, N. Engl. J. Med. 341 :556-62, 1999). HBV and HCV are the leading causes of liver cirrhosis and hepatocellular carcinoma. There are an estimated 1-1.2 million HIV infected persons in the U.S. (M. Glynn, P. Rhodes, Estimated HIV prevalence in the United States at the end of 2003, National HIV Prevention Conference, Atlanta, abstract (2005) 595) and 49 million more worldwide (Joint United Nations Program on HIV/AIDS, 2004, Global summary of the AIDS epidemic, WHO). Control of these three viral infections has been a major global health issue.

Serology assays and nucleic acid amplification tests (NAT) are available to diagnose and screen blood donors for these three viruses. Most of the blood supply in the U.S. is screened by serological tests for these three viruses and mini-pool (MP) NAT assays for HCV and HIV-I . A small percentage of the blood supply is currently tested using individual donation (ID) NAT for HCV and HIV-I (Coste et aL, Vox Sang. 88:289-303, 2005). Compared to the HBsAg test currently used to screen blood donors, the implementation of MP HBV NAT may marginally reduce the number of cases of transfusion-transmitted HBV (Coste et ah, Vox Sang. 88:289-303, 2005; Koppelrnan et al., Transfusion 45:1258-66, 2005; Kleinman et al., Transfusion 45:1247-57, 2005; Biswas et al., Transfusion 43:788-98, 2003; Stramer, Transfusion 45:1242-6, 2005). The development of NAT for the simultaneous detection of multiple pathogens (multiplex assays) could reduce the cost and increase the throughput needed for the implementation of ID NAT. Currently, there is only one FDA licensed duplex NAT assay for blood screening that can detect both HCV and HIV-I (Giachetti et ah, J. Clin. Microbiol. 40:2408-19, 2002), but it requires additional discriminatory assays to distinguish between the two viruses.

Microarray technology has been used to study the expression of thousands of genes in a single reaction. For example, microarrays can be used to detect bacteria at about 1000 cells/reaction (Theodore et al., John Hopkins APL Technical Digest. 25: 38-43, 2004). However, the utility of this technology for the simultaneous detection and discrimination of a set of DNA and RNA viruses (such as HBV, HCV and HIV-I), has not been developed.

Development of a cost-effective single-unit highly sensitive multiplex assay capable of detecting and discriminating HBV, HCV and HIV-I infections simultaneously could have an important impact in diagnosis and blood safety.

SUMMARY

Hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type-1 (HIV-I) are three transfusion transmitted viruses that have a major impact in blood safety and public health worldwide. The inventors have developed a sensitive and specific multiplex oligonucleotide array (such as a microarray) assay that can detect HBV, HCV, and HIV-I, for example in a human blood sample. The methods provided herein can be used to detect HBV, or HCV, or HΓV-1 , or two or all of these three viruses (for example simultaneously). For example, such methods can be used for diagnosis in clinics and in high risk groups, as discriminatory confirmation assay in screening of blood donors, and in research laboratories. Methods of detecting HBV, HCV, and HIV-I are provided. In particular examples, the method combines polymerase chain amplification (PCR) and microarray technologies. In one example, the method includes contacting nucleic acid molecules obtained from a subject with at least one oligonucleotide sequence that can specifically hybridize to an S-gene sequence of HBV (such as SEQ ID NO: 35, 36, 37, or 38), at least one oligonucleotide sequence that can specifically hybridize to a 5'-UTR sequence of HCV (such as SEQ ID NO: 31, 32, 33, or 34,), and at least one oligonucleotide sequence that can specifically hybridize to a gp41 gene sequence of HIV-I (such as SEQ ID NO: 27, 28, 29, or 30), under conditions that permit formation of oligonucleotide:nucleic acid molecule hybridization complexes. In particular examples, the nucleic acid molecules are also contacted with one or more control oligonucleotide sequences, such as one or more of those shown in SEQ ID NOS: 39, 40, 41, 42, 43 and 44. For example, the nucleic acid molecules (such as amplicons generated by multiplex PCR) can be contacted with an array that includes the desired oligonucleotide probes, and incubated under conditions that permit formation of oligonucleotide:nucleic acid molecule complexes. This can permit detection of HBV, HCV, and HIV-I in a sample simultaneously.

Subsequently, oligonucleotide:nucleic acid molecule complexes are detected. If oligonucleotide:nucleic acid molecule complexes form with one or more oligonucleotide sequences that can specifically hybridize to gp41 of HIV-I (such as SEQ ID NO: 27, 28, 29, or 30), this indicates that the subject (or the sample from where the nucleic acid molecules were obtained) is infected with HIV-I. If oligonucleotide:nucleic acid molecule complexes form with one or more oligonucleotide sequences that can specifically hybridize to a 5'-UTR sequence of HCV (such as SEQ ID NO: 31, 32, 33, or 34), this indicates that the subject (or the sample from where the nucleic acid molecules were obtained) is infected with HCV. If oligonucleotide:nucleic acid molecule complexes form with one or more oligonucleotide sequences that can specifically hybridize to an HBV S-gene sequence (such as SEQ ID NO: 35, 36, 37, or 38), this indicates that the subject (or the sample from where the nucleic acid molecules were obtained) is infected with HBV. In particular examples, the disclosed methods permit detection of at least 3 genomic equivalents (gEq) of HBV, at least 10 gEq of HCV, and at least 20 gEq of HIV-I . For example, in some examples the method can discriminate 3-10 gEq of HBV, 10-20 gEq of HCV, and 20 gEq of HIV-I. For example, if oligonucleotide:nucleic acid molecule complexes form with one or more oligonucleotide sequences that can specifically hybridize to gp41 of HIV-I (such as SEQ ID NO: 27, 28, 29, or 30), this can indicate that the subject (or the sample from where the nucleic acid molecules were obtained) is infected with at least 20 gEq of HIV-I . If oligonucleotide:nucleic acid molecule complexes form with one or more oligonucleotide sequences that can specifically hybridize to a 5'-UTR sequence of HCV (such as SEQ ID NO: 31, 32, 33, or 34), this can indicate that the subject (or the sample from where the nucleic acid molecules were obtained) is infected with at least 10 gEq of HCV, and wherein presence of oligonucleotide :nucleic acid molecule complexes form with one or more oligonucleotide sequences that can specifically hybridize to an HBV S-gene sequence (such as SEQ ID NO: 35, 36, 37, or 38), this can indicate that the subject (or the sample from where the nucleic acid molecules were obtained) is infected with at least 3 gEq of HBV.

In some examples, the method can further include treating an infection in subjects identified as infected with HBV, HCV, HIV-I, or combinations thereof. For example, such subjects can receive a treatment selected to avoid or reduce disease resulting from infection with HBV, HCV, HIV-I, or combinations thereof, such as administration of one or more antiviral agents.

The present disclosure also provides isolated nucleic acid molecules. Isolated nucleic acid primers that can be used to amplify an HBV, HCV, HIV- 1 , or a control sequence, are provided, such as those primers shown in SEQ ID NOS: 12- 26. Isolated nucleic acid probes that can specifically hybridize to HBV, HCV, HIV- 1, or control sequence are provided, such as those probes shown in SEQ ID NOS: 27-43. One skilled in the art will appreciate the minor changes can be made to these sequences without significantly affecting their ability to function as primers or probes. In some examples, such sequences include 1-3 nucleotide insertions, deletions, substitutions, or combinations thereof. Arrays are disclosed herein, such as DNA arrays. In some examples, the array includes isolated nucleic acid molecules (such as oligonucleotide probes) that permit detection of at least HBV, HCV, and HIV-I . For example, such arrays can have the ability to detect at least 3 gEq of HBV, at least 10 gEq of HCV, and at least 20 gEq of HIV-I . For example, in some examples the array can discriminate 3-10 gEq of HBV, 10-20 gEq of HCV, and 20 gEq of HIV-I. In some examples the array further includes isolated nucleic acid molecules that permit detection of control sequences. In a specific example, the array includes at least three isolated oligonucleotides, wherein at least one can detect HBV, at least one can detect HCV, and at least one can detect HIV-I. For example, the array can include at least one of SEQ ID NOS: 27-30, at least one of SEQ ID NOS: 31-34, and at least one of SEQ ID NOS: 35-38. In a particular example, the array includes or consists of SEQ ID NOS: 27-38 or SEQ ID NOS: 27-43.

The present disclosure also provides kits. In some examples, the kit includes at least one array of the present disclosure and a buffer solution in separate packaging. In some examples, the kit includes at least one array of the present disclosure and primers capable of amplification of an S-gene HBV sequence, primers capable of amplification of a 5'-UTR HCV sequence, and primers capable of amplification of a gp41 HIV-I sequence. For example, exemplary primers capable of amplifying an S-gene HBV sequence are shown in SEQ ID NOS: 20-23, exemplary primers capable of amplifying a 5'-UTR HCV sequence are shown in SEQ ID NOS: 16-19, and exemplary primers capable of amplifying a gp41 HIV-I sequence are shown in SEQ ID NOS: 12-15.

The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of genotyping 18 different HBV samples. Amino acid sequence of residues 40 to 90 of the S protein of HBV genotypes A, B, C, D, and E SEQ ID NOS: 7-1 1, respectively) deduced from nucleotide sequences in GenBank corresponding to accession numbers X02763, D00330, M12906, J02203, and X75657, respectively. The genotype-specific amino acids at positions no. 45, 46, 47, 49, 56, 57, 59, 64 and 85 are shown in bold and are underlined. Genotypes of the HBV samples were assigned by identifying the genotype-specific amino acids in each sample.

FIG. 2 A is a graph showing amplification of 10-fold dilutions of pAM-6 (a plasmid containing the HBV genome). Curves represent the normalized emission intensity of the reporter ΔRn versus the number of cycles.

FIG. 2B is a graph showing a standard curve for the quantitation of HBV copy number. The standard curve was constructed plotting the threshold cutoff values for each known amount of pAM-6 (filled dots). Values obtained from samples with unknown amounts of HBV DNA (open dots) were used to determine the HBV copy number.

FIG. 3 A is a schematic diagram showing the location of the oligonucleotides on the microarray.

FIG. 3B is a digital image showing that the quality control oligonucleotide probe (QCprb; SEQ ID NO: 43) on the array was detected by hybridization with Cy- 3 labeled antisense-quality control (antisense-QC; SEQ ID NO: 44) oligonucleotide, as a method to control the quality of each lot of array.

FIG.3C is a schematic diagram showing the location of arrays on slides. Sixteen sets (each set consists of two arrays for hybridization with one sample) were printed on one slide. Up to 16 samples could be tested on one slide.

FIGS. 4A-L are digital images showing microarray detection of HBV, HCV and HIV-I . Microarrays were hybridized with amplicons from multiplex PCR reactions labeled with Cy5 and antisense-QC oligonucleotide labeled with Cy3. Left panels show the hybridization of Cy5-labelled viral and internal control amplicons with the signature probes on the arrays scanned using laser light of 635 nm (red); right panels show the image of both Cy5-gene specific amplicons and Cy3- antisense-QC hybridized to the probes of the same sample scanned using double wave length: 625 nm and 532 nm (green). A. Normal serum (negative control). Only Cy5-labeled amplicons of 18S-rRNA gene hybridized with the internal control probes on the array. B-F. Samples from multiplex assay from samples containing HBV genotype A-E. B. WHO International Standard for HBV NAT (genotype A), at concentration of 1 International Unit (IU)/mL. C-F. HBV genotype B to E at 3- 10 gEq/reaction. Only HBV and internal control probes show signal. G. First WHO International Standard for HCV NAT (genotype 1) at 10 IU/mL. H. HCV from CBER HCV NAT Panel (genotype Ib), 10 gEq/reaction. I. HCV from NIBSC HCV Working Reagent (genotype 3) at 10 IU/mL. Only HCV and internal control probes show signals. J & K. Multiplex assay from samples containing HIV-I at 20 or 30 gEq/reaction. only HIV-I and internal control probes show signals. L. Simultaneous detection of HIV-I (30 gEq, group M, genotype B₃), HCV (20 gEq, genotype Ib), and HBV (10 gEq, genotype A) wherein all HIV-I₅ HCV, HBV and internal control probes show signals.

FIGS. 5A-B are digital images showing agarose gel electrophoresis analysis of PCR amplicons generated after two PCR reactions. A. Determination of the lower limit of detection (LOD) for HBV using WHO International Standard for HBV NAT as target. HBV band was negative at lane no. 3 (HBV concentration at 1 IU/mL), the same PCR product applied to a microarray containing HBV probes (SEQ ID NOS: 35-38) resulted in strong HBV signals (FIG. 4B). B. Simultaneous detection of HIV-I, HCV and HBV nucleic acids in one sample. The internal control band was undetectable on the gel. The same product as lane no. 6 was applied to a microarray containing probes (SEQ ID NOS: 27-42) and all three viruses and internal controls showed strong signals (FIG. 4L).

FIG. 6 is a digital image showing detection of HBV amplicons using gel electrophoresis or a microarray. Only five of the 10 HBV samples were detected on the gel, while 4 of these 5 negative on gel tested positive on microarray.

FIG. 7 is graph showing probit analysis for lower limit of detection (LOD) of WHO HBV based on the data of microarray multiplex assays of multiple duplicates of plasma containing 0, 1, 3.16, 10, and 31.6 IU/ml of WHO International Standard for HBV NAT (genotype A).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

SEQ ID NOS: 1-6 show primers used to amplify and sequence the S gene of HBV.

SEQ ID NOS: 7-11 show the amino acid sequence of residues 40 to 90 of the S protein of HBV genotypes A, B, C, D, and E, respectively.

SEQ ID NOS: 12-15 are PCR primer pairs, used to amplify a gp41 region of HIV-I.

SEQ ID NOS: 16-19 are PCR primer pairs used to amplify a region of the 5'UTR of HC V.

SEQ ID NOS: 20-23 are PCR primer pairs used to amplify a conserved region of the S gene of HBV.

SEQ ID NOS: 24-26 are PCR primers used to amplify a region of the human 18S rRNA gene.

SEQ ID NOS: 27-30 are nucleic acid probes used to detect HIV-I .

SEQ ID NOS: 31-34 are nucleic acid probes used to detect HCV.

SEQ ID NOS: 35-38 are nucleic acid probes used to detect HBV.

SEQ ID NOS: 39-42 are nucleic acid probes used to detect 18S rRNA gene.

SEQ ID NOS: 43-44 are nucleic acid quality control probes.

DETAILED DESCRIPTION Abbreviations and Terms

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular forms "a," "an," and "the" refer to one or more than one, unless the context clearly dictates otherwise. For example, the term "comprising a nucleic acid molecule" includes single or plural nucleic acids and is considered equivalent to the phrase "comprising at least one nucleic acid molecule." The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, "comprises" means "includes." Thus, "comprising A or B," means "including A, B, or A and B," without excluding additional elements. Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

5'-UTR 5'-untranlsated region gEq: genomic equivalents

HBV: hepatitis B virus

HCV: hepatitis C virus

HIV-I: human immunodeficiency virus type 1

NAT: nucleic acid amplification test

IU: international unit of virus assigned by WHO

5'-untranlsated region (5'-UTR): A non-coding region of HCV RNA that functions as an internal ribosomal entry site (IRES) and participates in the HCV genome translation within the cell to produce infective virions. The IRES directs viral protein translation via a cap-independent mechanism.

HCV 5'-UTR sequences are publicly available. For example, nucleotides 1 to about 341 of GenBank Accession No: M62321 (Aug. 2, 1993) and nucleotides 1 to about 329 of GenBank Accession No: D50480 (Feb. 10, 1999) disclose HCV 5'- UTR nucleic acid sequences. In one example, an HCV 5'-UTR sequence includes a full-length wild-type (or native) sequence, as well as HCV 5'-UTR allelic variants, variants, fragments, homologs or fusion sequences that retain the ability to direct viral protein translation. In certain examples, a HCV 5'-UTR has at least 80% sequence identity, for example at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to a native HCV 5'-UTR. In other examples, HCV 5'-UTR has a sequence that hybridizes under very high stringency conditions to nucleotides 1 to about 341 of GenBank Accession No: M62321 (Aug. 2, 1993), nucleotides 1 to about 329 of GenBank Accession No: D50480 (Feb. 10, 1999), nucleotides 1 to about 341 of GenBank Accession No: M67463 (Aug. 2, 1993), or nucleotides 1 to about 341 of GeπBank Accession No: D50480 (Aug. 9, 1997), and retains HCV 5'- UTR activity.

Administration: To provide or give a subject an agent by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. For example, a subject found to be infected with one or more of HBV, HCV, and HIV-I can be administered an appropriate therapeutic agent, such as an antiviral agent.

Amplifying a nucleic acid molecule: To increase the number of copies of a nucleic acid molecule, such as a gene or fragment of a gene, for example a region of an HBV, HCV, or HIV-I genome. The resulting amplification molecules are called amplification products or amplicons.

An example of in vitro amplification is the polymerase chain reaction (PCR), in which a biological sample obtained from a subject (such as a blood sample or fraction thereof, such as plasma) is contacted with a pair of oligonucleotide primers, under conditions that allow for hybridization of the primers to a target nucleic acid molecule in the sample (such as a target region of HBV, HCV, or HIV-I). The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid molecule. Other examples of in vitro amplification techniques include quantitative real-time PCR, strand displacement amplification (see USPN 5,744,311); transcription-free isothermal amplification (see USPN 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320 308); gap filling ligase chain reaction amplification (see USPN 5,427,930); coupled ligase detection and PCR (see USPN 6,027,889); NASBA™ RNA transcription-free amplification (see USPN 6,025,134), and multiplex PCR.

Array: An arrangement of molecules, such as biological macromolecules (such as nucleic acid molecules) or biological samples (such as tissue sections), in addressable locations on or in a substrate. A "microarray" is an array that is miniaturized so as to require or be aided by microscopic examination for evaluation or analysis. Arrays are sometimes called DNA chips or biochips. The array of molecules ("features") makes it possible to carry out a very large number of analyses on a sample at one time. In certain example arrays, one or more molecules (such as an oligonucleotide probe) will occur on the array a plurality of times (such as twice), for instance to provide internal controls. The number of addressable locations on the array can vary, for example from at least four, to at least 11, at least 12, at least 16, at least 18, at least 20, at least 30, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 500, least 550, at least 600, at least 800, at least 1000, at least 10,000, or more. In particular examples, an array includes nucleic acid molecules, such as oligonucleotide sequences that are at least 15 nucleotides in length, such as 15-60 or 30-57 nucleotides in length.

Within an array, each arrayed sample is addressable, in that its location can be reliably and consistently determined within at least two dimensions of the array. The feature application location on an array can assume different shapes. For example, the array can be regular (such as arranged in uniform rows and columns) or irregular. Thus, in ordered arrays the location of each sample is assigned to the sample at the time when it is applied to the array, and a key may be provided in order to correlate each location with the appropriate target or feature position. Often, ordered arrays are arranged in a symmetrical grid pattern, but samples can be arranged in other patterns (such as in radially distributed lines, spiral lines, or ordered clusters). Addressable arrays usually are computer readable, in that a computer can be programmed to correlate a particular address on the array with information about the sample at that position (such as hybridization or binding data, including for instance signal intensity). In some examples of computer readable formats, the individual features in the array are arranged regularly, for instance in a Cartesian grid pattern, which can be correlated to address information by a computer.

In particular examples an array includes oligonucleotide probes which can detect target sequences, such as target sequences from HBV, HCV, and HIV-I . For example, the array can include oligonucleotides capable of detecting the S-gene of HBV, the 5'-UTR of HCV, and gp41 of HIV-I, such as SEQ ID NOS: 35-38, 31-34, and 27-30, respectively. In a particular example, the array further includes control oligonucleotides, such as one or more of those shown in SEQ ID NOS: 39-44. Binding or stable binding: An association between two substances or molecules, such as the hybridization of one nucleic acid molecule to another. An oligonucleotide molecule binds or stably binds to a target nucleic acid molecule (such as the S-gene of HBV, the 5'-UTR of HCV, and gp41 of HIV-I) if a sufficient amount of the oligonucleotide molecule forms base pairs or is hybridized to its target nucleic acid molecule, to permit detection of that binding.

Binding can be detected by any procedure known to one skilled in the art, such as by physical or functional properties of the formed complexes, such as a target:oligonucleotide complex. For example, binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation, and the like.

Physical methods of detecting the binding of complementary strands of nucleic acid molecules, include but are not limited to, such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures. For example, one method involves observing a change in light absorption of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at 220 to 300 nm as the temperature is slowly increased. If the oligonucleotide or analog has bound to its target, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide (or analog) and target disassociate from each other, or melt. In another example, the method involves detecting a signal, such as a detectable label, present on one or both nucleic acid molecules.

In one example, the binding between an oligomer and its target nucleic acid is characterized by the temperature (T_m) at which 50% of the oligomer is melted from its target. A higher (T_m) means a stronger or more stable complex relative to a complex with a lower (T_m). cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences which determine transcription. cDNA can be synthesized by reverse transcription from messenger RNA extracted from cells.

Complementarity and percentage complementarity: Molecules with complementary nucleic acids form a stable duplex or triplex when the strands bind, (hybridize), to each other by forming Watson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable binding occurs when an oligonucleotide molecule remains detectably bound to a target nucleic acid sequence under the required conditions.

Complementarity is the degree to which bases in one nucleic acid strand base pair with the bases in a second nucleic acid strand. Complementarity is conveniently described by percentage, that is, the proportion of nucleotides that form base pairs between two strands or within a specific region or domain of two strands. For example, if 10 nucleotides of a 15-nucleotide oligonucleotide form base pairs with a targeted region of a DNA molecule, that oligonucleotide is said to have 66.67% complementarity to the region of DNA targeted.

In the present disclosure, "sufficient complementarity" means that a sufficient number of base pairs exist between an oligonucleotide molecule (such as SEQ ID NOS: 27-38) and a target nucleic acid sequence (such as a HBV S-gene, HCV 5'-UTR, or HIV-I gp41) to achieve detectable binding. When expressed or measured by percentage of base pairs formed, the percentage complementarity that fulfills this goal can range from as little as about 50% complementarity to full (100%) complementary. In general, sufficient complementarity is at least about 50%, for example at least about 75% complementarity, at least about 90% complementarity, at least about 95% complementarity, at least about 98% complementarity, or even at least about 100% complementarity.

A thorough treatment of the qualitative and quantitative considerations involved in establishing binding conditions that allow one skilled in the art to design appropriate oligonucleotides for use under the desired conditions is provided by Beltz et al. Methods Enzymol. 100:266-285, 1983, and by Sambrook et άl. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

DNA (deoxyribonucleic acid): A long chain polymer which includes the genetic material of most living organisms (some viruses have genes including ribonucleic acid, RNA). The repeating units in DNA polymers are four different nucleotides, each of which includes one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Genomic equivalent (gEq): A genomic equivalent is equal to one copy of DNA or cDNA detected by the quantitative PCR. Can include detection of complete virion particles and incomplete virion particles which only had DNA.

Glycoprotein 41 (gp41): A protein embedded in the outer envelope of HIV that mediates infection of HIV-I to CD4⁺ T cells by facilitating the fusion of the viral and cell membranes. The term gp41 includes any gp41 gene, cDNA, mRNA, or protein from any organism and that is a gp41 that can facilitate fusion of HIV-I to T cells. gp41 sequences are publicly available. For example, nucleotides 7304 to 8338 of GenBank Accession No: K03455 (Oct. 21, 2002) and GenBank Accession No: AF033819 (Aug. 28, 2002) disclose gp41 nucleic acid and protein sequences, respectively. In one example, a gp41 sequence includes a full-length wild-type (or native) sequence, as well as gp41 allelic variants, variants, fragments, homologs or fusion sequences that retain the ability to facilitate fusion of HIV-I to T cells. In certain examples, gp41 has at least 80% sequence identity, for example at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to a native gp41. In other examples, gp41 has a sequence that hybridizes under very high stringency conditions to nucleotides 7304 to 8338 of GenBank Accession No: K03455 (Oct. 21 , 2002), nucleotides 7304 to 8338 of GenBank Accession No. M 15654 (Aug. 2, 2006), or nucleotides 7304 to 8338 of GenBank Accession No. Zl 1530 (Nov. 14, 2006).

Hepatitis B virus (HBV): A DNA virus in the family Hepadnaviridae that causes hepatitis B. Includes all known serotypes (such as adr, adw, ayr, and ayw) based on antigenic epitopes present on the envelope proteins, and all known genotypes (such as A, B, C, D, E, F, G, H).

Hepatitis C virus (HCV): A small, enveloped, single-stranded, positive sense RNA virus in the family Flaviviridae, which causes hepatitis C. Includes all known genotypes (such as 1, 2, 3, 4, 5, and 6 and all subtypes within each genotype, and each quasispecies).

Human immunodeficiency virus type 1 (HIV-I): A DNA retrovirus that causes acquired immunodeficiency syndrome (AIDS). It is about 120 nm in diameter and roughly spherical. It includes two copies of positive single-stranded RNA that code for the virus's nine genes enclosed by a conical capsid. A matrix of the p27 viral protein surrounds the capsid, which is surrounded by the viral envelope. The envelope includes proteins from the host cell and the Env HIV protein (composed glycoprotein (gp) 120 and a stem of three gp41 molecules that anchor the structure into the viral envelope).

Hybridization: To form base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, NY (chapters 9 and 11). The following is an exemplary set of hybridization conditions and is not limiting:

Very High Stringency (detects sequences that share at least 90% identity)

Hybridization: 5x SSC at 65°C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65°C for 20 minutes each

High Stringency (detects sequences that share at least 80% identity) Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: 1 x SSC at 55°C-70°C for 30 minutes each

Low Stringency (detects sequences that share at least 50% identity)

Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

Isolated: An "isolated" biological component (such as a cell, nucleic acid molecule, protein) has been substantially separated or purified away from other biological components, for example in the cell in which the component naturally occurs, such as other cells, chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins. Therefore, an isolated nucleic acid molecule obtained from a blood sample includes nucleic acid molecules that are substantially separated away from other cellular components, such as proteins and organelles.

Label: An agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, or microscopy. For example, a label can be attached to a nucleic acid molecule thereby permitting detection of the nucleic acid molecule. Examples of labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).

Nucleic acid array: An arrangement of nucleic acids (such as DNA or RNA) in assigned locations on a matrix, such as that found in cDNA arrays, or oligonucleotide arrays.

Nucleic acid molecules: A deoxyribonucleotide or ribonucleotide polymer including, without limitation, cDNA, mRNA, genomic DNA, viral DNA, viral RNA, and synthetic (such as chemically synthesized) DNA. A nucleic acid molecule can be double-stranded or single-stranded. Where single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand.

The disclosed arrays can include isolated nucleic acid molecules that include specified lengths of a gp41 HIV sequence, an S-gene HBV sequence, and a 5'-UTR HCV nucleic acid sequence (or that can specifically hybridize to such sequences). Such molecules can include at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or at least 50 consecutive nucleotides of these sequences or more, and can be obtained from any region of a HIV, HBV₅ or HCV nucleic acid molecule.

Nucleotide: Includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

Oligonucleotide: A plurality of joined nucleotides joined by native phosphodiester bonds, for example between 6 and 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide.

Particular oligonucleotides and oligonucleotide analogs, such as those present on a nucleic acid array, can include linear sequences up to about 200 nucleotides in length (for example 10-200 nucleotides of an HBV, HCV, or HIV-I target nucleotide sequence, or a molecule that can specifically hybridize to such sequences), for example a sequence (such as DNA or RNA) that is at least 15 nucleotides, for example at least 18, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, or even at least 200 nucleotides long, such as 15 to 60 nucleotides, 30 to 60 nucleotides, or 30 to 57 nucleotides.

Oligonucleotide probe: An oligonucleotide that can be used to detect the presence of a complementary sequence by molecular hybridization. For example, an oligonucleotide probe can include a label that permits detection of oligonucleotide probe:target sequence hybridization complexes. In specific examples, an oligonucleotide probe includes 15 to 200 nucleotides, such as 30 to 57 nucleotides. Exemplary probes are provided in SEQ ID NOS: 27-43.

Primers: Short nucleic acid molecules, for instance DNA oligonucleotides 10 to 100 nucleotides in length, that can be annealed to a complementary target DNA strand (such as a HBV, HCV, or HIV-I target nucleic acid) by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand. Primer pairs can be used for amplification of a nucleic acid sequence, such as by PCR or other nucleic acid amplification methods known in the art. In particular examples a primer is 12 to 40 nucleotides in length, such as 19 to 30 nucleotides in lengths, for example 15, 19, 20, 25, 28 or 30 nucleotides in length. Exemplary primers are provided in SEQ ID NOS: 12-26.

Methods for preparing and using nucleic acid primers are described, for example, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).

Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified oligonucleotide preparation is one in which the oligonucleotide is more pure than in an environment including a complex mixture of oligonucleotides.

S-gene: A major protein constituent of the HBV envelope. The HBV envelope protein consists of three proteins, the large S protein (encoded by Pre-Sl, Pre-S2 and S gene), median S protein (encoded by pre S2 and S gene) and small S protein (encoded by S gene). The small S protein forms the major portion of envelope protein. The Pre-S2 gene (165 nucleotides) is located upstream of the S- gene. The term S-gene includes any S-gene sequence (such as gene, cDNA, mRNA, or protein) from any HBV virus.

S-gene sequences are publicly available. For example, nucleotides 155-835 of GenBank Accession No: X70185 (June 4, 1998) and nucleotides 157-837 of GenBank Accession No: VO 1460 (Jan. 28, 2003) disclose S-gene nucleic acid coding and protein sequences. In one example, a S-gene sequence includes a full- length wild-type (or native) sequence, as well as S-gene allelic variants, variants, fragments, homologs or fusion sequences that retain the ability to encode a functional HBV envelope protein. In certain examples, S-gene has at least 80% sequence identity, for example at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to a native S-gene. In other examples, S-gene has a sequence that hybridizes under very high stringency conditions to a sequence set forth in nucleotides 155 to 835 of GenBank Accession No. AF297624 (Oct.24, 2001) or GenBank Accession No. AB246337 (Oct. 28, 2006), and retains S-gene activity.

Sample: A biological specimen, such as those containing nucleic acid molecules (such as DNA or RNA), protein, or combinations thereof. Examples include, but are not limited to, peripheral blood, serum, plasma, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material. In one example, a sample includes peripheral blood or a fraction thereof, such as plasma or serum.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals (such as a veterinary subject).

Target sequence: A sequence of nucleotides located in a particular region in a genome (such as a region of HBV, HCV, or HIV-I) that corresponds to one or more specific sequences of interest. Examples of target sequences include a region of the S-gene of HBV, a region of the 5'-UTR of HCV, and a region of the gp41 gene of HIV-L

Therapeutically effective amount: An amount of a pharmaceutical preparation that alone, or together with a pharmaceutically acceptable carrier or one or more additional therapeutic agents, induces the desired response. A therapeutic agent, such as an antiviral agent, is administered in therapeutically effective amounts.

Effective amounts a therapeutic agent can be determined in many different ways, such as assaying for a reduction in viral load or improvement of physiological condition of a subject having an HBV, HCV, or HIV- 1 infection (such as increasing T-cell counts). Effective amounts also can be determined through various in vitro, in vivo or in situ assays.

Therapeutic agents can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of can be dependent on the source applied, the subject being treated, the severity and type of the condition being treated, and the manner of administration.

In one example, it is an amount sufficient to partially or completely alleviate symptoms of hepatitis B, hepatitis C, or AIDS in a subject. Treatment can involve only slowing the progression of the disease temporarily, but can also include halting or reversing the progression of the disease permanently. For example, a pharmaceutical preparation can decrease one or more symptoms of an infectious disease, for example decrease a symptom by at least 20%, at least 50%, at least 70%, at least 90%, at least 98%, or even at least 100%, as compared to an amount in the absence of the pharmaceutical preparation.

Treating a disease: "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such a sign or symptom of hepatitis B, hepatitis C, or AIDS. Treatment can also induce remission or cure of a condition, such as an infectious disease. In particular examples, treatment includes preventing a disease, for example by inhibiting the full development of a disease, such as preventing development of one or more symptoms of hepatitis B, hepatitis C, or AIDS. Prevention of a disease does not require a total absence of disease or symptoms. For example, a decrease of at least 20% or at least 50% can be sufficient.

Under conditions sufficient for: A phrase that is used to describe any environment that permits the desired activity.

In one example, includes incubating a nucleic acid molecule with an oligonucleotide probe under conditions (such as in an appropriate buffer, at an appropriate temperature and for an appropriate time) that permit hybridization of the probe to its target nucleic acid molecule.

In another example, includes administering a therapeutic agent to a subject in an amount and for a time sufficient to allow the desired activity. In particular examples, the desired activity is treating an HBV, HCV, or HIV-I infection.

Methods of Detecting HBV, HCV, and HIV-I

Transmission of HBV, HCV and HIV occurs primarily through exposure to infected blood. Before 1992, most infections occurred through blood transfusion or organ transplantation from infected donors, injection drug use, unsafe medical practices, occupational exposure, sex with an infected person, high risk sexual practices, intranasal cocaine use, use of blood products made from infected donors and others. Since the introduction of serological tests for HBV, HCV and HIV-I, the risk of transmission by blood transfusions or blood products decreased dramatically. However, there is still a small but significant transfusion transmission risk because these tests are still unable to detect the very early stages of infection (the pre-seroconversion "window period" infection where the viral load is very low and the immune response is beginning to develop) (Schreiber et al. , N. Engl. J. Med. 334:1685-90, 1996; Dodd et al, Transfusion 42:975-9, 2002).

Currently, there are several commercial nucleic acid amplification tests (NAT) assay systems that use different amplification methods, including PCR, transcription-mediated amplification (TMA), ligase chain reaction, and branched DNA (bDNA) signal amplification assay. Most commercial NAT assays detect each of these three viruses separately in pools of plasma samples. The sensitivities of these assays varies from below 50 to as many as 380 gEq/mL for HBV, <33 to 2855 gEq/mL for HCV, and 24 to 1000 gEq/mL for HIV-I (Adami et al, Clin. Microbiol. Infect. 10:1075-80, 2004; Cleland et al, Vox Sang. 81:93-101, 2001 ; Hammerle et al., Arch Virol. 142:1297-1306, 1997; Mercier et al., J. Virol. Meth. 77:1-9, 1999; Wozny et al, Viral Immunol. 10:73-82, 1997; Yerly et al., Transfusion 38:908-14, 1998; Zhao et al, Hepatobil Pancreat. Dis. Int. 2:234-41, 2003). There is only one NAT commercial product for blood screening capable of detecting HCV and HIV-I at a sensitivity of at least 100 gEq/mL for each virus (Giachetti et al., J. Clin. Microbiol. 40:2408-19, 2002). Most of the reported multiplex assays for HCV, HBV and/or HIV-I needed additional discrimination assays to determine the identity of the pathogen.

The current disclosure provides methods of detecting HBV₅ HCV₅ HIV-I, or combinations thereof (such as two or three of these). For example, the method permits detection of all three of HBV, HCV, and HIV-I, such as simultaneously. In particular examples, the method permits detection of a plurality of viruses including or consisting of HBV, HCV, and HIV-I. Such methods can be used to screen a sample, for example to determine if a patient is infected with one or more of these viruses, or to determine if a blood sample (for example in a blood bank) is infected with one or more of these viruses. The disclosed methods can permit detection and discrimination of two or three of these viruses substantially simultaneously in one reaction, such for example by using a nucleic acid (such as a DNA) array (for example an array that includes at least two probes specific for each of HIV-I, HBV and HCV, for example at least three or at least four probes). However, one skilled in the art will appreciate that separate reactions can be performed, for example in series or parallel. In particular examples, the disclosed methods do not require the use of additional discrimination assays or tests to determine the identity of the virus, that is, the identity of the virus can be determined directly using the disclosed methods (for example by detecting signal resulting from the formation of a hybridization complex). In particular examples, the disclosed methods can detect multiple genotypes for a given virus. It is shown herein that the disclosed microarray assay can detect five of the more common genotypes of HBV and three of the more common HCV genotypes.

In particular examples, the disclosed methods permit detection and discrimination with a sensitivity as low as 3-10 genome equivalents (gEq) of HBV, 10- 20 gEq of HCV, and 20 gEq of HIV-I per reaction, for example in a single multiplex reaction. Therefore, the method can in some examples permit detection of at least 3 gEq of HBV, at least 10 gEq of HCV, and at least 20 gEq of HIV-I, such as 3 gEq or less of HBV, 10 gEq or less of HCV, and 20 gEq or less of HIV-I . In particular examples, the 95% lower limit of detection (LOD) for HBV is 1.1 IU/mL (1 IU of HBV is equivalent to a mean of 5 gEq of HBV).

In particular examples, the disclosed methods include contacting nucleic acid molecules obtained from a subject with at least one oligonucleotide sequence (such as 1, 2, 3 or 4 sequences) that can specifically hybridize to an S-gene sequence of HBV, at least one oligonucleotide sequence (such as 1 , 2, 3 or 4 sequences) that can specifically hybridize to a 5'-UTR sequence of HCV, and at least one oligonucleotide sequence (such as 1, 2, 3 or 4 sequences) that can specifically hybridize to a gp41 gene sequence of HIV-I, under conditions that permit formation of oligonucleotide:nucleic acid molecule hybridization complexes (interchangeably referred to herein as oligonucleotidetnucleic acid molecule complexes). In particular examples, such oligonucleotide sequences are present on an array, such as immobilized to a glass slide. In such examples, the nucleic acid molecules from the subject are contacted with the array. For example, conserved regions of the S-gene sequence of HBV, 5'-UTR sequence of HCV, and HIV-I gp41 sequence can be used to design probes that will specifically hybridize to these conserved regions. In a specific example, such probes are 25-70 bp, such as 25-60 bp or 30-57 bp. In one example, partially overlapping gene specific oligonucleotide probes are provided for each virus (for example 2-8 of such probes, such as 3 or 4 of such probes), which decreases the likelihood that newly arising viral genotypes and mutants will be missed.

Contacting the nucleic acid molecules with the oligonucleotides that detect HBV, HCV, and HIV-I nucleic acid molecules can include incubating the nucleic acid molecules from the subject with the oligonucleotides for a sufficient time, under appropriate temperature and in an appropriate hybridization solution, to allow hybridization between the nucleic acid molecules and oligonucleotides, thereby forming complexes. Such reaction conditions can be routinely determined, for example based on the sequences of the oligonucleotides. Subsequently, the reaction can be analyzed to determine if any nucleic acid molecule:oligonucleotide complexes were formed, for example by detecting and quantifying the complexes (for example by detecting a signal from a label on the oligonucleotide or the nucleic acid molecule).

The method further includes detecting the oligonucleotide:nucleic acid molecule complexes (for example determining whether such complexes are present). In some examples, the oligonucleotide:nucleic acid molecule complexes are quantitated. For example, the presence of at least one (such as 1, 2, 3 or 4, for example at least 2, at least 3, or at least 4) oligonucleotidemucleic acid molecule complex containing an oligonucleotide sequence that can specifically hybridize to an HIV gp41 sequence of HIV-I indicates that the subject is infected with HIV-I, the presence of at least one (such as 1, 2, 3 or 4, for example at least 2, at least 3, or at least 4) oligonucleotidemucleic acid molecule complex containing an oligonucleotide sequence that can specifically hybridize to an HCV 5'-UTR sequence indicates that the subject is infected with HCV, and the presence of at least one (such as 1, 2, 3 or 4, for example at least 2, at least 3, or at least 4) oligonucleotide:nucleic acid molecule complex containing an oligonucleotide sequence that can specifically hybridize to an HBV S-gene sequence indicates that the subject is infected with HBV.

In a specific example, the presence of at least one (such as 1 , 2, 3 or 4, for example at least 2, at least 3, or at least 4) oligonucleotide:nucleic acid molecule complex containing an oligonucleotide sequence that can specifically hybridize to an HIV-I gp41 sequence indicates that the subject (or sample) is infected with at least 20 gEq of HIV-I, wherein the presence of at least one (such as 1, 2, 3 or 4, for example at least 2, at least 3, or at least 4) oligonucleotidemucleic acid molecule complex containing an oligonucleotide sequence that can specifically hybridize to an HCV 5'-UTR sequence indicates that the subject (or sample) is infected with at least 10 gEq (such as 10-20 gEq) of HCV, and wherein the presence of at least one (such as 1, 2, 3 or 4, for example at least 2, at least 3, or at least 4) oligonucleotide:nucleic acid molecule complex containing an oligonucleotide sequence that can specifically hybridize to an S-gene sequence of HBV indicates that the subject (or sample) is infected with at least 3 gEq of HBV.

Exemplary oligonucleotide sequences that can specifically hybridize to an HBV S-gene sequence are provided in SEQ ID NO: 35, 36, 37 and 38. Exemplary oligonucleotide sequences that can specifically hybridize to an HCV 5'-UTR sequence are provided in SEQ ID NO: 31, 32, 33, and 34. Exemplary oligonucleotide sequences that can specifically hybridize to an HIV-I gp41 sequence are provided in SEQ ID NO: 27, 28, 29, and 30. Therefore, the method can include incubating nucleic acid molecules obtained from a subject (or arnplicons thereof) with three or more of these sequences, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of SEQ ID NOS: 27-38.

In one example, nucleic acid molecules obtained from a subject are contacted with at least one (such as 1 , 2, 3 or 4) oligonucleotide sequence shown in SEQ ID NO: 27, 28, 29, or 30, at least one (such as 1, 2, 3 or 4) oligonucleotide sequence shown in SEQ ID NO: 31 , 32, 33, or 34, and at least one (such as 1 , 2, 3, or 4) oligonucleotide sequence shown in SEQ ID NO: 35, 36, 37, or 38 under conditions that permit formation of oligonucleotide.-nucleic acid molecule complexes. The presence of at least one (such as 1, 2, 3 or 4) oligonucleotide :nucleic acid molecule hybridization complex containing SEQ ID NO: 27, 28, 29, or 30 indicates that the subject is infected with HIV-I, the presence of at least one (such as 1, 2, 3 or 4) oligonucleotideinucleic acid molecule hybridization complex containing SEQ ID NO: 31, 32, 33, of 34 indicates that the subject is infected with HCV, and the presence of at least one (such as 1, 2, 3 or 4) oligonucleotide:nucleic acid molecule hybridization complex containing SEQ ID NO: 35, 36, 37, or 38 indicates that the subject is infected with HBV.

In a specific example, the nucleic acid molecules are contacted with the oligonucleotide sequences shown in SEQ ID NOS: 27-41 under conditions that permit formation of oligonucleotide:nucleic acid molecule complexes. In particular examples, the oligonucleotide sequences consist of SEQ ID NOS: 27-41, consist of SEQ ID NOS: 27-41 and 43, or consist of SEQ ID NOS: 27-43. The presence of oligonucleotide:nucleic acid molecule complexes with SEQ ID NO: 27, 28, 29, or 30 (such as 1, 2, 3 or 4 of these sequences) indicates that the subject is infected with HIV-I, the presence of oligonucleotidemucleic acid molecule complexes with SEQ ID NO: 31, 32, 33, or 34 (such as 1, 2, 3 or 4 of these sequences) indicates that the subject is infected with HCV, and the presence of oligonucleotide:nucleic acid molecule complexes with SEQ ID NO: 35, 36, 37, or 38 (such as 1, 2, 3, or 4 of these sequences) indicates that the subject is infected with HBV.

Clinical Samples

The nucleic acid molecules obtained from a subject (such as DNA or RNA) can be screened using the disclosed methods to detect HBV, HCV, and HIV-I nucleic acid molecules. Appropriate specimens for use with the current disclosure in diagnosing HIV-I, HBV, and HCV infection include any conventional clinical samples that include nucleic acids, for instance blood or blood-fractions (such as plasma). For example, a sample from a subject can include HBV, HCV, or HIV-I nucleic acids, if the subject is infected with that particular virus.

Techniques for acquisition of such samples are well known in the art. Plasma or other blood fractions can be prepared in the conventional manner. For example, about 250 μL to 2 ml of plasma or serum can be used for the extraction of DNA or RNA for use in amplification reactions. Once a sample has been obtained, the sample can be used directly, concentrated (for example by centrifugation or filtration), purified, amplified, or combinations thereof. For example, nucleic acid molecules can isolated from a sample obtained from the subject using routine methods known in the art. For example, DNA and RNA can be obtained using a commercially available kit (such as the NucliSens isolation kit, Organon Teknika, Netherlands and the High Pure viral nucleic acid kit, Roche Diagnostics, Mannheim, Germany). In one example, the DNA or RNA preparation method yields a nucleotide preparation that is accessible to, and amenable to, nucleic acid amplification. Additional methods are provided in the Examples below.

Amplification of nucleic acid molecules

In some examples, nucleic acid molecules obtained from a subject are amplified prior to analysis using the disclosed methods. In such examples the resulting nucleic acid amplicons are analyzed using the disclosed methods. Any nucleic acid amplification method can be used, such as PCR amplification. The disclosure is not limited to particular amplification methods. If desired, multiple rounds of amplification can be performed.

Amplicons can include a label, such as a fluorophore. Such labels can be attached to the amplicon during or after synthesis of the amplicon, for example by primer extension thermocycling. Methods of labeling nucleic acid molecules are known in the art.

In particular examples, the method includes amplification of targeted HBV, HCV, and HIV-I sequences (such as target S-gene, 5'-UTR, and gp41 sequences, respectively) by multiplex PCR. For example, multiplex PCR primers, such as those provided in SEQ ID NOS: 12-23, can be used.

For example, the method can include obtaining a blood sample from the subject, isolating nucleic acid molecules from the blood sample, amplifying the nucleic acid molecules in a first PCR reaction, thereby generating a first set of amplicons, and amplifying the first set of amplicons in a second PCR reaction, thereby generating a second set of amplicons, wherein the second set of amplicons is contacted with the oligonucleotide sequences, under conditions that permit formation of oligonucleotideinucleic acid molecule complexes.

Control sequences

In particular examples, the nucleic acid molecules to be analyzed to determine if HIV-I, HCV₃ and HBV nucleic acid molecules are present, are further contacted with one or more control oligonucleotide sequences.

For example, nucleic acid molecules to be analyzed can include one or more sequences known to be present or absent from the nucleic acid sample. In one example, the nucleic acid molecules to be analyzed include at least one known nucleic acid sequence, such as a human 18S ribosome RNA (rKN A) sequence. Such sequences can be used to confirm proper functioning of a PCR reaction and proper functioning of the array. For example, using the primers in SEQ ID NOS: 24-26 generates amplicons from 18S rRNA. If the appropriate amplicons are not generated, this indicates that the PCR reaction is not working properly. Such amplicons (which can include a label), when present in the nucleic acid sample, will hybridize to appropriate oligonucleotide sequences on an array (such as one or more of SEQ ID NOS: 39-41), thereby generating detectable hybridization complexes. If such hybridization complexes are not detected, this indicates that the hybridization did not work properly, and the results should be discarded. In particular examples, such control sequences do not significantly interfere with the ability to detect HIV-I, HBV, or HCV sequences.

Additional control nucleic acid sequences are provided in SEQ ID NOS: 43 and 44. For example, SEQ ID NO: 44 provides an antisense quality control sequence (that can include a label), that will hybridize under highly stringent conditions to the quality control probe shown in SEQ ID NO: 43 (which can be immobilized to an array).

Detecting Oligonucleotide :Tar get Nucleic Acid Molecule Complexes

The nucleic acid molecules obtained from the subject (for example from a blood sample) can contain HCV, HBV, or HIV-I (or combinations thereof) target nucleic acid molecules, if the subject is infected with these viruses. Such viral nucleic acid molecules can be detected to determine if the subject (or blood obtained from the subject) is infected with one or more of these three viruses. The present disclosure is not limited to particular methods of detection. Any method of detecting a nucleic acid molecule can be used, such as physical or functional assays.

Methods for labeling nucleic acid molecules so that they can be detected are well known. Labels include molecules that permit detection of the nucleic acid molecule to which they are attached. Examples of such labels include non- radiolabels and radiolabels. Non-radiolabels include, but are not limited to enzymes, chemiluminescent compounds, fluorophores, metal complexes, haptens, colorimetric agents, dyes, or combinations thereof. Radiolabels include, but are not limited to, ¹²⁵I and ³⁵S. Radioactive and fluorescent labeling methods, as well as other methods known in the art, are suitable for use with the present disclosure. In one example, primers used to amplify the subject's nucleic acids are labeled (such as with biotin, a radiolabel, or a fluorophore), thereby permitting labeling of the resulting amplicons. In another example, the resulting amplicons are end-labeled (for example with one or more fluorophores) to form labeled amplified material. In a specific example, amplified nucleic acid molecules can be labeled by including labeled nucleotides in the amplification reactions. A subject's labeled nucleic acid molecules (or amplicons) can be applied to an array containing oligonucleotides.

The nucleic acid molecules obtained from the subject (or amplicons generated from such nucleic acid molecules) that may include viral nucleic acid molecules if the subject is infected with HBV, HCV, HIV-I, or combinations thereof, are applied to an oligonucleotide array under suitable hybridization conditions to form a hybridization complex between oligonucleotides on the array and nucleic acid molecules from the subject (referred to as oligonucleotide :nucleic acid molecule complexes or hybridization complexes). In particular examples, the nucleic acid molecules include a label, and the hybridization complexes are detected by detecting the label. In one example, a pre-treatment solution of organic compounds, solutions that include organic compounds, or hot water, can be applied before hybridization (see U.S. Patent No. 5,985,567).

Hybridization conditions for a given combination of array and target material can be optimized routinely in an empirical manner close to the T_m of the expected duplexes, thereby maximizing the discriminating power of the method. Identification of the location in the array, such as a cell, in which binding occurs, permits a rapid and accurate identification of target nucleic acid sequences (see below).

The hybridization conditions are selected to permit discrimination between matched and mismatched oligonucleotides. Hybridization conditions can be chosen to correspond to those known to be suitable in standard procedures for hybridization to filters and then optimized for use with the arrays of the disclosure. For example, conditions suitable for hybridization of one type of target would be adjusted for the use of other targets for the array. In particular, temperature is controlled to substantially eliminate formation of duplexes between sequences other than exactly complementary sequences. A variety of known hybridization solvents can be employed, the choice being dependent on considerations known to one of skill in the art (see U.S. Patent 5,981,185).

Once the nucleic acid molecules from the subject have been allowed to hybridize with the oligonucleotides present in the array, the presence of the hybridization complex can be analyzed, for example by detecting the complexes. Detecting a hybridized complex in an array of oligonucleotide probes has been previously described (see U.S. Patent No. 5,985,567). In one example, detection includes detecting one or more labels present on the oligonucleotides, the nucleic acid molecules obtained from the subject (or amplicons therefrom), or both. In particular examples, developing includes applying a buffer. In one example, the buffer is sodium saline citrate, sodium saline phosphate, tetramethylammonium chloride, sodium saline citrate in ethylenediaminetetra-acetic, sodium saline citrate in sodium dodecyl sulfate, sodium saline phosphate in ethylenediaminetetra-acetic, sodium saline phosphate in sodium dodecyl sulfate, tetramethylammonium chloride in ethylenediaminetetra-acetic, tetramethylammonium chloride in sodium dodecyl sulfate, or combinations thereof. However, other suitable buffer solutions can also be used.

Detection can further include treating the hybridized complex with a conjugating solution to effect conjugation or coupling of the hybridized complex with the detection label, and treating the conjugated, hybridized complex with a detection reagent. In one example, the conjugating solution includes streptavidin alkaline phosphatase, avidin alkaline phosphatase, or horseradish peroxidase. Specific, non-limiting examples of conjugating solutions include streptavidin alkaline phosphatase, avidin alkaline phosphatase, or horseradish peroxidase. The conjugated, hybridized complex can be treated with a detection reagent. In one example, the detection reagent includes enzyme-labeled fluorescence reagents or calorimetric reagents. In one specific non-limiting example, the detection reagent is enzyme-labeled fluorescence reagent (ELF) from Molecular Probes, Inc. (Eugene, OR). The hybridized complex can then be placed on a detection device, such as an ultraviolet (UV) transilluminator (manufactured by UVP, Inc. of Upland, CA). The signal is developed and the increased signal intensity can be recorded with a recording device, such as a charge coupled device (CCD) camera (manufactured by Photometries, Inc. of Tucson, AZ). In particular examples, these steps are not performed when fluorophores or radiolabels are used.

The method can further include quantification, for instance by determining an absolute or relative amount of hybridization or number of hybridization complexes formed. In particular examples, an increase of least 2-fold (such as at least 5-fold, at least 10-fold, or at least 100-fold) in the amount of hybridization as compared to a reference amount of hybridization, indicates that the subject is infected with HBV, HCV, HIV-I , or combinations thereof. For example, a reference amount can be an amount of hybridization detected or expected from a nucleic acid sample known to not include HBV, HCV, or HIV-I nucleic acid molecules.

Methods of Treating HBV, HCV, and HIV-I

Subjects identified as infected with HBV, HCV, HlV-I, or combinations thereof, using the methods provided herein can be selected for treatment. For example, subjects having an HBV, HCV, or HIV-I infection (or combinations thereof) can be treated with therapeutically effective amounts of an appropriate agent, such as one or more antiviral agents, immune system modulators, protease inhibitors (such as VX 950), polymerase inhibitors, or combinations thereof. Such treatment protocols, including appropriate dosages and administration regimens, are well known to those skilled in the art.

For example, a subject infected with HBV (such as a subject having hepatitis

B) can be administered therapeutically effective amounts of one or more antivirals (such as lamivudine, adefovir, telbivudine, and Entecavir), immune system modulators (such as interferon alpha and pegylated interferon alpha), or combinations thereof.

For example, a subject infected with HCV (such as a subject having hepatitis

C) can be administered therapeutically effective amounts of one or more antivirals (such as ribavirin), immune system modulators (such as interferon alpha, pegylated interferon alpha, and immunoglobulins against HCV), or combinations thereof.

For example, a subject infected with HIV-I (such as a subject having AIDS) can be administered therapeutically effective amounts of one or more anti-retroviral agents (such as "cocktails" of two or more of nucleoside analogue reverse transcriptase inhibitors (NARTIs or NRTIs), protease inhibitors, and non-nucleoside reverse transcriptase inhibitors (NNRTI)).

Isolated Nucleic Acid Molecules

The present disclosure provides isolated nucleic acid molecules. Isolated nucleic acid molecules include primers that can be used to amplify a target sequence, or probes that can hybridize to a target sequence. Exemplary target sequences include a region of the S-gene of HBV, a region of the 5'-UTR of HCV, and a region of gp41 of HIV-I, such as highly conserved regions. When it is said herein that an isolated nucleic acid molecule consists of a particular SEQ ID NO: this does not exclude the possibility that the isolated nucleic acid molecule also includes a label (such as a fluorophore).

Isolated nucleic acid primers that can be used to amplify an HBV, HCV, HIV-I, or a control sequence, are disclosed. In a specific example, the primers permit amplification (for example using multiplex PCR) of a region of the S-gene of HBV, a region of the 5'-UTR of HCV, or a region of gp41 of HIV-I . Exemplary primers that can amplify a region of the S-gene of HBV are provided in SEQ ID NOS: 20-23, exemplary primers that can amplify a region of the 5'-UTR of HCV are provided in SEQ ID NOS: 16-19, and exemplary primers that can amplify a region of gp41 of HIV-I are provided in SEQ ID NOS: 12-15. Also provided are isolated nucleic acid primers that function as control primers, for example to amplify a control target sequence known to be present in the sample, for example to confirm that the amplification reaction is working properly. Exemplary control primers are provided in SEQ ID NOS: 24-26. Therefore, the present disclosure provides an isolated nucleic acid molecule consisting of any of SEQ ID NOS: 12-26.

Isolated nucleic acid probes that can specifically hybridize to HBV, HCV₅ HIV-I, or control sequence are provided. In a specific example, the probes can hybridize to a region of the S-gene of HBV, a region of the 5'-UTR of HCV₅ or a region of gp41 of HIV-I . Exemplary probes that can hybridize to a region of the S- gene of HBV are provided in SEQ ID NOS: 39-42, exemplary probes that can hybridize to a region of the 5'-UTR of HCV are provided in SEQ ID NOS: 31-34, and exemplary probes that can hybridize to a region of gp41 of HIV-I are provided in SEQ ID NOS: 27-30. Also provided are isolated nucleic acid probes that function as control probes, for example to confirm that the assay is working properly. Exemplary control probes are provided in SEQ ID NOS: 43-44. Therefore, the present disclosure provides an isolated nucleic acid molecule consisting of any of SEQ ID NOS: 27-44.

One skilled in the art will appreciate the minor changes can be made to the particularly disclosed isolated nucleic acid probe and primer sequences without significantly affecting their ability to function as primers or probes. In one example, any isolated nucleic acid molecule shown in SEQ ID NO: 12-44 includes 1-3 nucleotide insertions, deletions, substitutions, or combinations thereof.

In a specific example, the isolated nucleic acid molecules shown in SEQ ID NOS: 12-44 includes a deletion of 1-3 nucleotides (such as 1, 2, or 3 nucleotide deletions). The nucleotide deletions can occur anywhere in SEQ ID NOS: 12-44, since such a small deletion in these isolated nucleic acid molecules will not significantly affect the ability of these molecules to hybridize to their respective target sequence. Therefore, the present disclosure provides an isolated nucleic acid molecule consisting of any of SEQ ID NOS: 12-44 having 1-3 nucleotide deletions. In a specific example, any of the isolated nucleic acid molecules shown in SEQ ID NOS: 12-44 includes 1-2 nucleotide substitutions (such as 1 or 2 nucleotide substitutions). The substitutions can occur anywhere in SEQ ID NOS: 12-44, since such a small number of substitutions in these isolated nucleic acid molecules will not significantly affect the ability of these molecules to hybridize to their respective target sequence. Therefore, the present disclosure provides an isolated nucleic acid molecule consisting of any of SEQ ID NOS: 27-44 having 1-2 nucleotide substitutions.

In a specific example, the isolated nucleic acid molecules shown in SEQ ID NOS: 27-44 includes 3 or more nucleotide substitutions (such as 3 nucleotide substitutions). The substitutions can occur at the end of SEQ ID NOS: 27-44 (such as anywhere in the first 5 nucleotides or the last 5 nucleotides), since such a small number of substitutions at the ends of these isolated nucleic acid molecules will not significantly affect the ability of these molecules to hybridize to their respective target sequence. In some examples where there are three or more nucleotide substitutions, such substitutions are not evenly distributed in the target sequence, as such substitutions would significantly affect the ability of these molecules to hybridize to their respective target sequence. Therefore, the present disclosure provides an isolated nucleic acid molecule consisting of any of SEQ ID NOS: 27-44 having 1-3 nucleotide substitutions (such as anywhere in the first 5 nucleotides or the last Snucleotides).

Arrays

Arrays are disclosed herein, such as DNA arrays. In some examples, the array includes isolated nucleic acid molecules (such as oligonucleotide probes) that permit detection of at least HBV, HCV, and HIV-I, for example in a blood sample. In some examples the array further includes isolated nucleic acid molecules that permit detection of control sequences.

In a specific example, the array includes at least three isolated oligonucleotides, wherein at least one oligonucleotide can detect HBV, at least one can detect HCV, and at least one can detect HIV-I . In one example, the array includes any combination of at least three of the probes shown in SEQ ID NOS: 27- 38, such as at least one of SEQ ID NOS: 27-30, at least one of SEQ ID NOS: 31-34, and at least one of SEQ ID NOS: 35-38. In another example, the array includes as at least two (at least three, or all four) of SEQ ID NOS: 27-30, at least two (at least three, or all four) of SEQ ID NOS: 31-34, and at least two (at least three, or all four) of SEQ ID NOS: 35-38. In a particular example, the array includes or consists of SEQ ID NOS: 27-38 or SEQ ID NOS: 27-43. In a particular example, the array includes or consists of SEQ ID NOS: 27-41, and 43.

The array can include additional oligonucleotide probes, such as control probes to detect the presence of a control nucleic acid molecule (for example any of SEQ ID NOS: 39-43).

In one example, oligonucleotide probes are attached to the surface of a solid support for use in detecting at least HBV, HCV, and HIV-I, such as in a sample obtained from a subject. The oligonucleotide probes bound to the array can specifically bind nucleic acid molecules obtained from the subject, or nucleic acid molecules amplified from the subject (such as under high stringency conditions). Thus, sequences of use with the method are oligonucleotide probes that recognize HBV, HCV, or HIV-I nucleic acid sequences. Such sequences can be determined by examining the sequences of HBV, HCV, and HIV-I, and choosing oligonucleotide sequences that specifically anneal to a particular region of HBV, HCV, or HIV-I, but not others.

The methods and apparatus in accordance with the present disclosure takes advantage of the fact that under appropriate conditions oligonucleotides form base- paired duplexes with nucleic acid molecules that have a complementary base sequence. The stability of the duplex is dependent on a number of factors, including the length of the oligonucleotides, the base composition, and the composition of the solution in which hybridization is effected. The effects of base composition on duplex stability can be reduced by carrying out the hybridization in particular solutions, for example in the presence of high concentrations of tertiary or quaternary amines.

The thermal stability of the duplex is also dependent on the degree of sequence similarity between the sequences. By carrying out the hybridization at temperatures lower than the anticipated T_m's of the type of duplexes expected to be formed between the target sequences and the oligonucleotides bound to the array, the rate of formation of mis-matched duplexes may be substantially reduced.

The length of each oligonucleotide sequence employed in the array can be selected to optimize binding of target HBV, HCV₅ and HIV-I nucleic acid sequences. An optimum length for use with a particular HBV, HCV, and HIV-I target nucleic acid sequence under specific screening conditions can be determined empirically. Thus, the length for each individual element of the set of oligonucleotide sequences included in the array can be optimized for screening. In one example, oligonucleotide probes are from about 25 to about 50 nucleotides in length, such as about 30 to about 57 nucleotides in length.

The oligonucleotide probe sequences forming the array can be directly linked to the support. Alternatively, the oligonucleotide probes can be attached to the support by spacers or linkers. Methods of attaching nucleic acid molecules to a solid support are well-known in the art. For example, oligonucleotide probes can be immobilized on a silylated glass slide.

Array substrate

The solid support can be formed from an organic polymer. Suitable materials for the solid support include, but are not limited to: polypropylene, polyethylene, polybutylene, polyisobutylene, polybutadiene, polyisoprene, polyvinylpyrrolidine, polytetrafluroethylene, polyvinylidene difluroide, polyfluoroethylene-propylene, polyethylenevinyl alcohol, polymethylpentene, polycholorotrifluoroethylene, polysulfomes, hydroxylated biaxially oriented polypropylene, aminated biaxially oriented polypropylene, thiolated biaxially oriented polypropylene, etyleneacrylic acid, thylene methacrylic acid, and blends of copolymers thereof (see U.S. Patent No. 5,985,567, herein incorporated by reference).

In general, suitable characteristics of the material that can be used to form the solid support surface include: being amenable to surface activation such that upon activation, the surface of the support is capable of covalently attaching a biomolecule such as an oligonucleotide thereto; amenability to "in situ" synthesis of biomolecules; being chemically inert such that at the areas on the support not occupied by the oligonucleotides are not amenable to non-specific binding, or when non-specific binding occurs, such materials can be readily removed from the surface without removing the oligonucleotides.

^■ In one example, the solid support surface is polypropylene. Polypropylene is chemically inert and hydrophobic. Non-specific binding is generally avoidable, and detection sensitivity is improved. Polypropylene has good chemical resistance to a variety of organic acids (such as formic acid), organic agents (such as acetone or ethanol), bases (such as sodium hydroxide), salts (such as sodium chloride), oxidizing agents (such as peracetic acid), and mineral acids (such as hydrochloric acid). Polypropylene also provides a low fluorescence background, which minimizes background interference and increases the sensitivity of the signal of interest.

In another example, a surface activated organic polymer is used as the solid support surface. One example of a surface activated organic polymer is a polypropylene material aminated via radio frequency plasma discharge. Such materials are easily utilized for the attachment of nucleotide molecules. The amine groups on the activated organic polymers are reactive with nucleotide molecules such that the nucleotide molecules can be bound to the polymers. Other reactive groups can also be used, such as carboxylated, hydroxylated, thiolated, or active ester groups.

Array formats

A wide variety of array formats can be employed in accordance with the present disclosure. One example includes a linear array of oligonucleotide bands, generally referred to in the art as a dipstick. Another suitable format includes a two- dimensional pattern of discrete cells (such as 4096 squares in a 64 by 64 array). As is appreciated by those skilled in the art, other array formats including, but not limited to slot (rectangular) and circular arrays are equally suitable for use (see U.S. Patent No. 5,981,185, herein incorporated by reference). In one example, the array is formed on a polymer medium, which is a thread, membrane or film. An example of an organic polymer medium is a polypropylene sheet having a thickness on the order of about 1 mil. (0.001 inch) to about 20 mil., although the thickness of the film is not critical and can be varied over a fairly broad range. Particularly disclosed for preparation of arrays at this time are biaxially oriented polypropylene (BOPP) films; in addition to their durability, BOPP films exhibit a low background fluorescence.

The array formats of the present disclosure can be included in a variety of different types of formats. A "format" includes any format to which the solid support can be affixed, such as microtiter plates, test tubes, inorganic sheets, dipsticks, and the like. For example, when the solid support is a polypropylene thread, one or more polypropylene threads can be affixed to a plastic dipstick-type device; polypropylene membranes can be affixed to glass slides. The particular format is, in and of itself, unimportant. All that is necessary is that the solid support can be affixed thereto without affecting the functional behavior of the solid support or any biopolymer absorbed thereon, and that the format (such as the dipstick or slide) is stable to any materials into which the device is introduced (such as clinical samples and hybridization solutions).

The arrays of the present disclosure can be prepared by a variety of approaches. In one example, oligonucleotide sequences (such as one or more of those shown in SEQ ID NOS: 27-44, such as at least 2, at least 3, or all of these sequences) are synthesized separately and then attached to a solid support (see U.S. Patent No. 6,013,789). In another example, sequences are synthesized directly onto the support to provide the desired array (see U.S. Patent No. 5,554,501). Suitable methods for covalently coupling oligonucleotides to a solid support and for directly synthesizing the oligonucleotides onto the support are known to those working in the field; a summary of suitable methods can be found in Matson et al., Anal. Biochem. 217:306-10, 1994. In one example, the oligonucleotides are synthesized onto the support using conventional chemical techniques for preparing oligonucleotides on solid supports (such as see PCT applications WO 85/01051 and WO 89/10977, or U.S. Patent No. 5,554,501).

A suitable array can be produced using automated means to synthesize oligonucleotides in the cells of the array by laying down the precursors for the four bases in a predetermined pattern. Briefly, a multiple-channel automated chemical delivery system is employed to create oligonucleotide probe populations in parallel rows (corresponding in number to the number of channels in the delivery system) across the substrate. Following completion of oligonucleotide synthesis in a first direction, the substrate can then be rotated by 90° to permit synthesis to proceed within a second (2°) set of rows that are now perpendicular to the first set. This process creates a multiple-channel array whose intersection generates a plurality of discrete cells.

The oligonucleotides can be bound to the polypropylene support by either the 3' end of the oligonucleotide or by the 5' end of the oligonucleotide. In one example, the oligonucleotides are bound to the solid support by the 3¹ end. However, one of skill in the art can determine whether the use of the 3' end or the 5¹ end of the oligonucleotide is suitable for bonding to the solid support. In general, the internal complementarity of an oligonucleotide probe in the region of the 3¹ end and the 5¹ end determines binding to the support.

In particular examples, the oligonucleotide probes on the array include one or more labels, that permit detection of oligonucleotide probe:target sequence hybridization complexes.

Kits

The present disclosure also provides kits that can be used to detect HBV, HCV, and HIV-I in a sample, such as a blood sample. Such kits can be used with the methods of the present disclosure. In some examples, the kit includes at least one array of the present disclosure. In some examples, the kit includes at least 2 probes or primers provided herein, such as 2 or more of SEQ ID NOS: 12-44. The kits can further include a one or more of a buffer solution, a conjugating solution for developing the signal of interest, or a detection reagent for detecting the signal of interest, each in separate packaging, such as a container. Exemplary buffers include hybridization buffers, wash buffers, PCR buffers, and the like.

In some examples, the kit includes probes capable of detecting an HBV S- gene, probes capable of detecting a 5'-UTR of HCV, and probes capable of detecting a gp41 of HIV-I. Such probes can be attached to a solid support to form an array. For example, exemplary probes capable of detecting an S-gene HBV sequence are shown in SEQ ID NOS: 35-38, exemplary probes capable of detecting a 5'-UTR HCV sequence are shown in SEQ ID NOS: 31-34, and exemplary probes capable of detecting a gp41 HIV-I sequence are shown in SEQ ID NOS: 27-30. Therefore, in some examples a kit includes or consists of two or more of SEQ ID NOS: 27-38, such as four or more of SEQ ID NOS: 27-38, six or more of SEQ ID NOS: 27-38, or all of SEQ ID NOS: 27-38. _^

In some examples, the kit includes primers capable of amplifying a region of the HBV S-gene, primers capable of amplifying a region of the 5'-UTR HCV sequence, and primers capable of amplifying a region of the gp41 HIV-I sequence. Such kits can further include at least one array of the present disclosure. For example, exemplary primers capable of amplifying an S-gene HBV sequence are shown in SEQ ID NOS: 20-23, exemplary primers capable of amplifying a 5'-UTR HCV sequence are shown in SEQ ID NOS: 16-19, and exemplary primers capable of amplifying a gp41 HIV-I sequence are shown in SEQ ID NOS: 12-15. Therefore, in some examples a kit includes or consists of two or more of SEQ ID NOS: 12-23, such as four or more of SEQ ID NOS: 12-23, six or more of SEQ ID NOS: 12-23, or all of SEQ ID NOS: 12-23. In a specific example, a kit includes at least one array of the present disclosure and one or more of SEQ ID NOS: 12-23, such as two or more of SEQ ID NOS: 12-23, six or more of SEQ ID NOS: 12-23, or all of SEQ ID NOS: 12-23.

Example 1 Genotyping and Determination of HBV Copy Number

This example describes methods used to determine the genotype and copy number of HBV in clinical samples using a microarray. The genotypes of HBV samples were determined by sequencing the S gene and identification of residues in the amino acid sequences that are shared with known HBV genotypes.

HBV samples (n=18) included the WHO NAT International Standard for HBV DNA assays (obtained from National Institute for Biological Standard and Control, NIBSC, code no. 97/746, contains IxIO⁶ IU, South Mimms, UK), 11 plasma samples of HBV seroconversion panels collected from patients with acute HBV infection (ZeptoMatrix Corp. Franklin, MA), and sera samples from 3 HBV chronic carriers and 3 HBsAg- negative HBV-DNA(+) hepatocellular carcinoma (HCC) patients (Cancer Institute/Hospital, Beijing, China). HBV DNA was extracted from human plasma by phenol-chloroform-isoamyl alcohol (Invitrogen Corp.) after proteinase K digestion (Hsia et al., J. Med. Virol. 70:20-6, 2003).

A fragment of S gene of HBV (241 bp) was amplified from the isolated HBV DNA by nested PCR and sequenced using the primers in Table 1 (SEQ ID NOS: 1- 6) in an automated sequencer (ABI Prism 310 Genetic Analyzer, Applied Biosystems, Foster City, CA). The deduced amino acid sequences were aligned with the consensus sequences of known genotypes of HBV in GenBank using MacVector 7.2 software (Genetic Computer Group, Madison, WI). HBV genotypes were determined by the type-specific amino acids: 45, 46, 47, 49, 56, 57, 59, 64, and 85 of the S gene (see Hsia et al, J, Med. Virol. 70:20-6, 2003 and Hsia et al, Transfusion. 46:1829-35, 2006).

The deduced amino acid sequences of the HBV samples compared to consensus sequences of different genotypes of HBV is shown in FIG. 1 (SEQ ID NOS: 7-11). The identified genotypes of the samples are listed in Table 2.

Real-time quantitative PCR using SYBR Green I was used to assess the copy numbers of HBV DNA in all the HBV samples since it allows the detection of various genotypes of HBV using one pair of primers (Papin et al. , J. CHn. Microbiol. 42: 1511-8, 2004). Real time PCR primers were targeted to the conserved region of the S gene of HBV (Table 1, SEQ ID NOS: 5 and 6). To quantify the copy number of the HBV samples, a standard curve was constructed using 10-fold serial dilutions (10¹ tolO⁵ gEq/reaction) of an HBV plasmid-pAM-6 containing the full genome of HBV (adw subtype, genotype A) (ATCC, Rockville, MD) (Hsia et al., Transfusion. 46:1829-35, 2006). The PCR reaction included 12.5 μl of 2X SYBR Green PCR master mix (Applied Biosystems, Foster City, CA), 2.0 μl of primers (SEQ ID NOS: 5 and 6; 300 nM each), 0.5 μl of DEPC water, and 10 μl of DNA sample. Amplification was performed on an ABI Prism 7700 Sequence Detector using conditions suggested by the manufacturer (Applied Biosystems). Standard curves were generated and copy numbers of unknown samples were calculated using the Sequence Detector v.1.6.3. software (Applied Biosystems). The specificity of the amplification was confirmed by analysis of T_m dissociation curves following the real time PCR. As shown in FIG. 2B, a linear correlation was observed between the HBV plasmid copy number and the threshold cutoff with a coefficient of 0.997. This standard curve was used to determine the copy number of the 18 HBV DNA samples, which varied from 2.2x10² to 2.3xl0⁹copies/mL (Table 2).

Example 2 Multiplex Detection and Discrimination of HBV, HCV and HIV-I

This example describes methods used to detect HBV, HCV, and HIV-I in blood samples, using a single microarray.

Isolation and amplification of nucleic acid molecules

HBV-containing samples used are those described in Example 1. Three plasma samples from chronic HCV patients including (1) First WHO International Standard for HCV NAT (NIBSC code no. 97/790 (genotype 1); (2) CBER HCV RNA Panel (genotype Ib); (3) NIBSC HCV Working Reagent for NAT (code no.78/576). HIV-I sample(group M, subtype B) was from the CBER HIV-I RNA panel. Unknown samples may contain either viral RNA or DNA, both RNA (from HCV or/and HIV-I) and DNA (from HBV) can be extracted with NucliSens kit from human plasma or sera.

HIV-I and HCV RNA was extracted from human plasma or sera with LS Trizol (Invitrogen, Corp., Carlsbad, CA), precipitated with isopropanol in the presence of glycogen (30 μg/mL). cDNA of HCV or HIV-I was synthesized using Superscript II reverse-transcriptase and oligo (dT)i₂-i8 or viral specific primers (Invitrogen Corp.) at 42°C for 50 minutes. HBV DNA was extracted from human plasma as described in Example 1. cDNA of HCV or HIV-I and HBV DNA were amplified on a 9600 or 9700 GeneAmp PCR System (PerkinElmer Life Sciences, Inc., Boston, MA) by two rounds of PCR. As an internal control to monitor the assay process, human cellular DNA extracted from whole blood cells was included in each reaction (10 ng/reaction) starting from the extraction procedure and used as template to amplify the 18 S ribosomal internal control PCR fragments. PCR primer pairs of 19-28 nucleotides in length and T_n, of 58-60 ⁰C, which did not form dimers and hairpins, were used to amplify conserved region of S gene of HBV, 5¹UTR of HCV or gp41 of HIV-I (see Table 3, SEQ ID NOS: 20-23, 16- 19, and 12-15, respectively). Primers for amplifying a region of human 18S rRNA are shown in SEQ ID NOS: 24-26. Primers were selected using Mac Vector 7.2 (Genetic Computer group, Madison, WI) and Primer 3 ' software (Selvapandiyan et al., J. MoI. Diagn. 7:268-75, 2005). The specificity of viral primers and lack of cross-hybridization with sequences from human or other organisms was demonstrated using the BLAST program.

The first PCR reaction included a primer mix of four pairs of primers (SED ID NO. 12, 13, 16,17, 20, 21, 24, and 25) and AmpliTaq Gold 0.5 μl, buffer, MgCl₂ and GeneAmp 10 mM dNTP mix (Applied Biosystems, Foster City, CA) and nucleic acid template in 50 μl volume. The second PCR used primer mix of four pairs of primers (SEQ ID NOS: 14, 15, 18, 19, 22, 23, 24, and 26) and same reagents as in the first PCR. The concentrations of the individual primers for the first and the second PCR reactions were: HBV forward and reverse primers 400 nM each, HCV forward and reverse primers 600 nM each, HIV-I forward and reverse primers 900 nM each, and internal control forward and reverse primers 100 nM each. The PCR program for both rounds of PCR was: 95°C 10 minutes (activate the Taq enzyme); followed by 40 cycles of 95°C 15 seconds, 50⁰C 15 seconds, 68°C 45 seconds; and a final extension of 10 minutes at 72° C.

Labeling of Single Stranded DNA (ssDNA)

The resulting PCR products (amplicons) were purified with MinElute PCR purification Kit (Qiagen, GmbH, Germany) and then labeled with indodicarbocyanine (Cy5) by primer extension thermocycling in a multiplex reaction in the presence of reverse primers and Cy5-dCTP (PerkinElmer Life Sciences Inc., Boston, MA). The labeling reaction used a primer mix of HIV reverse primer (SEQ ID NO: 15: 400 nM), HCV reverse primer (SEQ ID NO: 19: 400 nM), HBV reverse primer (SEQ ID NO: 23; 900 nM), Internal control reverse primer (SEQ ID NO. 26: 10OnM), 1 μl of a mixture containing dATP, dGTP, dTTP (5 mM each), 1 μl of dCTP (1 mM), and 1 μl of Cynine 5-dCTP (1 mM, Perkin Elmer, Boston, MA), the purified second PCR products described above, in 50 μl reaction using the same enzyme and PCR conditions as above. After labeling, the labeled amplicons were purified with the MinElute purification Kit according to the manufacturer's instructions.

A synthetic antisense quality control oligonucleotide (antisense-QC; SEQ ID NO: 44), with sequences complementary to the quality control oligonucleotide probe (QCprb) (SEQ ID NO: 43) (Table 4), was labeled with indocarbocynine (Cy3)-dCTP at the 5'-end during synthesis. The Cy3 -anti sense QC was purified by high performance liquid chromatography (HPLC) (CBER, FDA).

Microarray

The microarray probes were designed as follows. Amplicons of each specific gene (HIV-I, HCV, HBV, and internal control A or B) was targeted by four gene specific probes (Table 4, SEQ ID NOS: 27-30, 31-34, 35-38, and 39-42, respectively). Each probe was 30-57 nucleotides in length with T_m of 65-73⁰C. A schematic diagram of the array is shown in FIG. 3 A. Twenty probes, four for each pathogen and the internal control, were used.

The microarray was generated as follows. To monitor the performance of the spotting procedure, each oligonucleotide probe was spiked with an equal volume of 1/10 dilution of the quality control probe (QCprb; SEQ ID NO: 43). Microarrays were printed on silylated glass slides (SSC aldehyde-slide, CEL Associates Inc., Pearland, Texas) using a contact microspotting robot (Cartesian Technologies, Inc.) and a ChipMaker microspotting device with a single CMP -7 pin (TeleChem International Inc.). Each slide was spotted with 16 sets of arrays (each set consists of 2 arrays, wherein each array consists of 20 spots, as shown in FIGS. 3A and 3 C). Humidity in the microspotting chamber was maintained at 70-75% to ensure the efficient coupling of amino-modified oligonucleotide probes with the aldehyde groups on the glass surface. The slides were then treated as described previously (Chizhikov et al., J. Clin. Microbiol. 40:2398-407, 2002). The quality of each lot of printed slides was tested by hybridizing the last printed slide with Cy3-anti-QC (SEQ ID NO: 44) (FIG. 3B). Slides were dried and stored at -7O⁰C for 6-8 months.

Microarray hybridization and scanning

Immediately before hybridization, the Cy5-labeled amplicons (3.3 μl) and Cy3-labeled anti-sense QC (SEQ ID NO: 44) (0.2 μl) were mixed with an equal volume of 2X hybridization buffer (10X Denhardt solution, 12X SSC, 0.2% Tween 20) in a final volume of 7 μl, and denatured by incubating 1 minute at 95°C and 1 minute on ice. The hybridization mixture was applied to one set of microarrays (consists of two microarrays) and covered with a plastic cover slide. Since each slide was printed with 16 sets of microarrays, 16 samples could be applied per slide. The slides were placed in hybridization chambers and incubated in a 45⁰C water bath for 1.5 hour. After incubation, slides were washed sequentially in 6X SSC containing 0.2% Tween 20, 6X SSC, and 2X SSC for 1 minute each and dried at room temperature in a hood (2 minutes) or centrifuge for 2 minutes at 1500 rpm.

The slides were scanned on a GenePix 4000B Microarray Scanner (Molecular Devices Corp, Axon Instruments, Sunnyvale, CA), a laser-excitation- based epi fluorescence scanning system at 635 nm (red) and 635/532 nm (red and green) to detect Cy5 and Cy5 with Cy3, respectively. The data was analyzed with GenPro Pix 5.1 software (Molecular Devices Corp, Axon Instruments).

Results

A sample was reactive (positive for a particular virus) if at least one of the four probes for a given virus showed a positive signal. The microarray detected HBV genotype A at 1 IU/mL, genotype B, C, and D at 3 gEq/reaction (FIGS. 4B-E) and HBV genotype E at 10 gEq/reaction (FIG. 4F). The microarrays also detected HCV genotype 1 and 3 at 10 IU/mL and genotype Ib at 10-20 gEq/reaction (FIGS. 4G, 4H, 41) and HIV-I at 20 or 30 gEq/reaction (FIGS. 4J and 4K).

These results demonstrate that the primers (SEQ ID NOS: 12-23) and probes (SEQ ID NOS: 27-38) are highly specific; cross-hybridization was not detected in the microarray. More over, amplification of negative plasma and sera samples did not result in detectable viral signal in the microarray (FIG. 4A). In addition, the microarray detected three (10 gEq HBV, 20 gEq HCV and 30 gEq HIV-I) (FIG. 4L) viruses present in one sample.

For HBV, the 95% lower limit of detection is 1.1 IWmL, and 50% lower limit of detection is 0.86 IU/mL (1 IU of HBV is equivalent to a mean of 5 gEq). The specificity is estimated as at least 99.9%

Example 3 Comparing Detection Sensitivity of Microarray and Gel Electrophoresis

To compare the sensitivity of detecting the ampKcons by microarray hybridization versus agarose gel electrophoresis, the same PCR products were analyzed in both systems to determine the lower limit of detection of HBV.

WHO International Standard for HBV NAT (10⁶ IU/mL, NIBSC code no. 97/746) was serially diluted with normal human plasma from one patient who tested negative for HCV, HBV, HIV-I by serological and NAT assay (Blood bank NIH, Bethesda, MD) to 31.6, 10, 3.16, 1 IU/mL. Three replicates of 31.6 IU/ml, 10 replicates for each of 10 IU/mL, 3.16 IU/ml and 1 IU/mL (with addition of 10 ng human DNA as internal control in each sample) and normal plasma were extracted with NucliSens kit. The nucleic acids were subjected to two rounds of multiplex PCR reaction (40 cycles each, 50 μl volume) using the pairs of primers (SEQ ID NOS: 12-26) as described above. The resulting second PCR amplicons were analyzed by gel electrophoresis and microarray. The results data were analyzed by probit analysis using a program provided by Division of Biostatistics (CBER, FDA, Rockville, MD), analyzed using SAS software.

Part of the gel electrophoresis analysis is shown in FIG. 5 A. At HBV 1 IU/ml, no HBV band is detectable on lane no. 3. In contrast, the same PCR product applied to the microarray shows a strong signal of HBV and internal control (FIG. 4B). When three viruses were present in one sample, the gel analysis only detects the viral bands, no internal control band was detected (FIG. 5 B). In contrast, when the same PCR product as lane no. 6 was applied to the microarray, all the three virus and internal control produced strong signals (FIG. 4L). As shown in FIG. 6, at plasma concentration of 1 IU/mL₅ an HBV band was detected on the agarose gel in only 5/10 samples, but HBV was detected in 9/10 samples using the microarray. HBV samples that tested positive on the gel were all positive on microarray (two of five are shown in FIG. 6). Therefore, at an HBV concentration of 1 IU/mL, the positivity rate on the gel was 5/10, while the positivity rate on the microarray was 9/10 (Table 5).

Table 5: Microarray Multiplex Assay for WHO HBV International Standard

Probit analysis of results demonstrated that the 95% lower limit of detection of HBV by microarray is I.I IU/mL (FIG. 7), while it is 15.9 IU/mL for gel analysis. Therefore, the disclosed microarray method is at least 14.5 -fold more sensitive than gel analysis.

These results demonstrate that microarray hybridization detected amplicons not observed by gel electrophoresis. The sensitivity of the microarray assay is at least 14.5-fold higher than agarose gel electrophoresis analysis.

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated examples are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims. TABLE 1. Primers for DNA sequencing and quantitative real time PCR

Function Target 5' to 3' Sequence (SEQ ID NO:) Target Gene Positions (nt)^* Amplicons (bp) and name

PCR & sequencing HBV 1St PCR S21(F) ATCAGGATTCCTAGGACCC (1) S 169-187

S22(R) AGGACAAACGGGCAACATAC (2) S 459-478 310

2nd PCR S23(F) GCGGGGTTTTTCTTGTTGAC (3) S 203-222 S24 (R) AACCAACAAGAAGATGAGGC (4) S 423-443 241

Real time PCR HBV Taq-S-11(F) CTGTGCCTTGGGTGGCTTT (5) S 182-200 Taq-S-12(R) AAGGAAAGAAGTCAGAAGGCAAAA (6) S 249-272 91

* The 5' T residue of the unique EcoRl site in the positive strand of HBV is considered base number 1.

Kind of patients Code No. Source HBsAg Genotype HBV DNA gEq /mL

Chronic HBV

1 ^• WHO International HBV Standard + A 5.4x10⁶

2 Serum from HBV chronic carrier #1 + C 2.3x10⁹

3 Serum from HBV chronic carrier #2 + C 1.5x10⁹

4 Serum from HBV chronic carrier #3 + C 1.7x10⁸

Acute HBV

5 S-C Panel #1^a, member #21 + A 9.3x10²

6 S-C Panel #2, member #17 + E 4.0x10⁵

7 S-C Panel #3, member #11 + A 1.9x10⁴

8 S-C Panel #4, member #8 + D 6.0x10²

9 S-C Panel #5, member #7 + A 2.1x10³

10 S-C Panel #6, member #12 + A 5.7x10⁴

11 S-C Panel #7, member #14 + A 1.1x10⁴

12 S-C Panel #8, member #16 + A 1.6x10⁴

13 S-C Panel #9, member #16 + A 1.1x10⁴

14 S-C Panel #10, member #11 + D 2.2x10⁴

15 S-C Panel #11, member #11 + A 1.4x10⁴

Sera from HBsAg (-) HBV DNA (+) hepatocellular carcinoma

16 HBV associated HCC #1^b B 3.5x10³

17 HBV associated HCC #2^b B 2.0x10³

18 HBV associated HCC #3° C 2.2x10²

a S-C Panel: Sero-conversion panel b HBsAg was tested by Auszyme, Abbott Laboratories, IL, CH. c HBsAg was tested by AxSym, Abbott Laboratories, IL, CH.

Viral specific primers

1^st PCR IV-105 (F) HIV TCπAGGAGCAGCAGGAAGCACTATGGG (12)^" 7789-7816

IV-106 ( R) AACGACAAAGGTGAGTATCCCTGCCTM¹ (13) GP⁴¹ 8347-8374 586 2^nd PCR IV-35 (F) TCCTGGGGATTTGGGGTTG (14) 7999-8017

IV-36(R ) CπGCTGGTTTTGCGATTC (15) 8166-8184 186

1^s' PCR CV- IOI (F) HCV TCCCCTGTGAGGAACTACTGTCTTC (16) 41-65

CV-102 (R) TTGAGGTTTAGGATTCGTGCTC (17) 5'-UTR 344-365 325 2^nd PCR CV-21 (F) ATGGCGTTAGTATGAGTGTCGTRC (18) 85-108

CV-22(R) CCCAAATCTCCAGGCATTGAG (19) 211-231 147

1StPCR S-61 (F) HBV ATCTTCCTGCTGGTGGCTC (20) Pre-S 51-69

S-62 (R) AAACGGGCAACATACCTTG (21) 455-473 423

4- 2¹^ PCR BV-H (F) CAGTCCCCAACCTCCMTCAC (22) S 314-334

BV-12 (R) GGCATAGCAGCAGGATGAAGAG (23) 404-425 112

Internal control primers i st PCR Hu-5 (F) Human gene TCGAAGACGATCAGATACCGT (24) 1147-1167 Hu-4 (R) TTGCAACCATACTCCCCCCG (25) 18S rRNA 1264-1283 137

2^nd PCR Hu-5 (F) Same as above Hu-6 ( R) ATACTCCCCCCGGAACC (26)

1259-1275 130

* Numbering according to sequences in GenBank accession nos. K03455 (HIV-I), M62321 (HCV Ia), X70185 (HBV adw2), and M10098 (human 18S rRNA). The 5' T residue of the unique EcoRI site in the positive strand of HBV is considered base number 1. The 1st nucleotide of the 18S ribosome RNA gene is consi base number 1.

TABLE 4. Viral specific, internal control, and quality control oligonucleotide probes

Target Probe Sequence (SEQ ID NO:) Gene Positions T_m(°C) (nt)*

HIV 1 AACTCATTTGCACCACTGCTGTGCCTTGGAATGCT (27) 8026-8060 68 2 GCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCAC (28) 8058-8099 65 3 ACGACCTGGATGGAGTGGGACAGAGAAATTAACAA (29) gp ,^'41 8100-8134 65 4 GTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGMGAATC (30; 8114-8165 65

HCV 1 TGCAGCCTCCAGGACCCCCCCTCCCGGGAG (31) 106-136 73 2 CTCCCGGGAGAGCCATAGTGGTCTGCGGAACCG (32) 126-158 68 3 GAACCGGTGAGTACACCGGAATTGCCAGGACGACC (33) 5'-UTR 153-187 68 4 GGACGACCGGGTCCTTTCTTGGATCAACCCGCTC (34) 180-213 71

HBV 1 TCACCAACCTCCTGTCCTCCAATTTGTCCTGGTTAT (35) 335-370 66

O 2 TGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTG (36) 347-381 66 3 TGTCCTGGTTATCGCTGGATGTGTCTGCGG (37) S-gene 359-388 66 4 TCGCTGGATGTGTCTGCGGCGTTTTATCATATTCC (38) 370-404 66

Internal 1 GTCGTAGTTCCGACCATAAACGATGCCGACCGG (39) 1166-1198 67

Control 2 GGCGATGCGGCGGCGTTATTCCCATGACCC (40) 18S rRNA 1197-1226 69

3 CCGCCGGGCAGCTTCCGGGAAACCAAAGTCTTTG (41) 1225-1258 71

4t TCGAAGACGATCAGATACCGTCGTAGTTCCGACC (42)

1147-1180 67

Quality Control Probe (QCprb) TTGGCAGAAGCTATGAAACGATATGGG (43) NA 82 Antisense-QC CCCATATCGTTTCATAGCTTCTGCCAA (44) NA 82

* Refer the numbering to Table 1 footnote. ^fAn anti-primer probe that is an additional control for the presence of primers in reaction mix.

Claims

We claim:

1. A method of detecting a plurality of viruses comprising hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency type I virus (HIV-I), comprising: contacting nucleic acid molecules obtained from a subject with at least one oligonucleotide sequence shown in SEQ ID NO: 27, 28, 29, or 30, at least one oligonucleotide sequence shown in SEQ ID NO: 31 , 32, 33, or 34, and at least one oligonucleotide sequence shown in SEQ ID NO: 35, 36, 37, or 38 under conditions that permit formation of oligonucleotide:nucleic acid molecule complexes; and determining whether oligonucleotide:nucleic acid molecule complexes are present, wherein presence of oligonucleotide:nucleic acid molecule complexes with SEQ ID NO: 27, 28, 29, or 30 indicates that the subject is infected with HIV-I, wherein presence of oligonucleotide:nucleic acid molecule complexes with SEQ ID NO: 31, 32, 33, or 34 indicates that the subject is infected with HCV, and wherein presence of oligonucleotide:nucleic acid molecule complexes with SEQ ID NO: 35, 36, 37 or 38 indicates that the subject is infected with HBV.

2. A method of detecting at least 3 genomic equivalents (gEq) of HBV, at least 10 gEq of HCV, and at least 20 gEq of HIV-I, comprising: contacting nucleic acid molecules obtained from a subject with at least one oligonucleotide sequence that can specifically hybridize to an S-gene sequence of HBV₅ at least one oligonucleotide sequence that can specifically hybridize to a 5'- UTR sequence of HCV, and at least one oligonucleotide sequence that can specifically hybridize to a gp41 gene sequence of HIV-I, under conditions that permit formation of oligonucleotide:nucleic acid molecule complexes, and detecting the oligonucleotide:nucleic acid molecule complexes, wherein presence of nucleic acid molecule complexes with at least one oligonucleotide sequence that can specifically hybridize to a gp41 gene sequence of HIV-I indicates that the subject is infected with at least 20 gEq of HIV-I, wherein presence of nucleic acid molecule complexes with at least one oligonucleotide sequence that can specifically hybridize to a 5'-UTR sequence of HCV indicates that the subject is infected with at least 10 gEq of HCV, and wherein presence of oligonucleotide:nucleic acid molecule complexes with at least one oligonucleotide sequence that can specifically hybridize to an S-gene sequence of HBV indicates that the subject is infected with at least 3 gEq of HBV.

3. The method of claim 1 or 2, wherein the nucleic acid molecules obtained from the subject comprise nucleic acid amplicons.

4. The method of claim 3, wherein the amplicons are produced by PCR amplification of the nucleic acid molecules obtained from the subject.

5. The method of claim 1 or 2, wherein the nucleic acid molecules are further contacted with one or more control oligonucleotide sequences under conditions that permit formation of control oligonμcleotide:nucleic acid molecule complexes.

6. The method of claim 5, wherein the one or more control oligonucleotide comprises one or more of the oligonucleotide sequences shown in SEQ ID NOS: 39, 40, 41, 42, 43 or 44.

7. The method of claim 1 or 2, wherein detecting the oligonucleotide :nucleic acid molecule complexes comprises quantitating the oligonucleotide:nucleic acid molecule complexes.

8. The method of claim 1 or 2, wherein detecting the oligonucleotide :nucleic acid molecule complexes comprises detecting a label associated with the oligonucleotidemucleic acid molecule complexes.

9. The method of claim 8, wherein the label comprises a fluorophore.

10. The method of claim 8, wherein the label is associated with the nucleic acid molecules.

11. The method of claim 10, further comprising labeling the nucleic acid molecules obtained from the subject with a label.

12. The method of claim 1, wherein the method can discriminate at least 3 genomic equivalents (gEq) of HBV, at least 10 gEq of HCV, and at least 20 gEq of HIV-I .

13. The method of claim 1 or 2, wherein the method can discriminate 3-10 gEq of HBV, 10-20 gEq of HCV, and 20 gEq of HIV-I .

14. The method of claim 1, wherein the nucleic acid molecules are contacted with the oligonucleotide sequences shown in SEQ ID NOS: 27-41 under conditions that permit formation of oligonucleotidernucleic acid molecule complexes, and wherein presence of oligonucleotide:nucleic acid molecule complexes with SEQ ID NO: 27, 28, 29, or 30 indicates that the subject is infected with HIV-I, wherein presence of oligonucleotidernucleic acid molecule complexes with SEQ ID NO: 31, 32, 33, or 34 indicates that the subject is infected with HCV, and wherein presence of oligonucleotide:nucleic acid molecule complexes with SEQ ID NO: 35, 36, 37, or 38 indicates that the subject is infected with HBV.

15. The method of claim 14, wherein presence of oligonucleotide:nucleic acid molecule complexes with at least two of SEQ ID NO: 27, 28, 29, or 30 indicates that the subject is infected with HIV-I, wherein presence of oligonucleotide:nucleic acid molecule complexes with at least two of SEQ ID NO: 31, 32, 33, or 34 indicates that the subject is infected with HCV, and wherein presence of oligonucleotidernucleic acid molecule complexes with at least two of SEQ ID NO: 35, 36, 37, or 38 indicates that the subject is infected with HBV.

16. The method of claim 14, wherein the oligonucleotide sequences consist of SEQ ID NOS: 27-41.

17. The method of claim 14, wherein the oligonucleotide sequences consist of SEQ ID NOS: 27-41 and 43.

18. The method of claim 14, wherein the oligonucleotide sequences consist of SEQ ID NOS: 27-43.

19. The method of claim 1 or 2, wherein the oligonucleotide sequences are present on an array substrate and the nucleic acid molecules are contacted with the array.

20. The method of claim 1 or 2, wherein the nucleic acid molecules are isolated from a sample obtained from the subject.

21. The method of claim 20, where the nucleic acid molecules comprise mKNA or cDNA.

22. The method of claim 1 or 2, wherein contacting the nucleic acid molecules with the oligonucleotides comprises: incubating the nucleic acid molecules from the subject with the oligonucleotides for a time sufficient to allow hybridization between the nucleic acid molecules and oligonucleotides, thereby forming nucleic acid molecule:oligonucleotide complexes; and analyzing the nucleic acid molecule:oligonucleotide complexes to determine if nucleic acid molecule:oligonucleotide complexes were formed, wherein the presence of nucleic acid molecule:oligonucleotide complexes indicates the subject is infected with HBV, HCV, HIV-I, or combinations thereof.

23. The method of claim 22, wherein analyzing the nucleic acid molecule:oligonucleotide complexes comprises quantitating an amount of nucleic acid hybridization, and wherein an increase of least 2-fold in the amount of hybridization as compared to a reference amount of hybridization, indicates that the subject is infected with HBV, HCV, HIV-I, or combinations thereof.

24. The method of claim 23, wherein the reference amount is an amount of hybridization present from a nucleic acid sample from a subject not infected with HBV, HCV, or HIV-I.

25. The method of claim 22, wherein analyzing the nucleic acid molecule:oligonucleotide complexes includes detecting and quantifying the complexes.

26. The method of claim I₅ further comprising: obtaining a blood sample from the subject; isolating nucleic acid molecules from the blood sample; amplifying the nucleic acid molecules in a first PCR reaction, thereby generating a first set of amplicons; and amplifying the first set of amplicons in a second PCR reaction, thereby generating a second set of amplicons, wherein the second set of amplicons labeled with a fluorophore is contacted with the oligonucleotide sequences under conditions that permit formation of oligonucleotide:nucleic acid molecule complexes.

27. The method of claim 1 or 2, further comprising administering to a subject determined to be infected with HBV, HCV, HIV-I, or combinations thereof, a treatment to avoid or reduce disease resulting from infection with HBV, HCV, HIV- 1, or combinations thereof.

28. The method of claim 27, wherein the treatment comprises treating the subject with a therapeutic amount of an antiviral agent.

29. A method of determining if a subject is infected with HBV, HCV, or HIV-I, comprising: applying nucleic acid molecule amplicons generated from a sample obtained from the subject to an array, wherein the array comprises the oligonucleotide sequences shown in SEQ ID NOS: 27-41 and 43; incubating the amplicons with the array for a time sufficient to allow hybridization between the amplicons and oligonucleotide sequences, thereby forming ampliconroligonucleotide complexes; and detecting the amplicon:oligonucleotide complexes to determine if the subject is infected with HBV, HCV, or HIV-I, wherein the presence of amplicon:oligonucleotide complexes indicates that the subject is infected with one or more of HBV, HCV, and HIV-I .

30. An isolated nucleic acid molecule consisting of:

(a) any of SEQ ID NOS: 12-26; or

(b) any of SEQ ID NOS: 27-44.

31. An array, comprising: at least three isolated nucleic acid molecules of claim 30(b) attached to an array substrate.

32. The array of claim 31, wherein the at least three isolated nucleic acid molecules comprise: at least one of SEQ ID NOS: 27-30; at least one of SEQ ID NOS: 31 -34; and at least one of SEQ ID NOS: 35-38.

33. The array of claim 32, wherein the at least three isolated nucleic acid molecules comprise SEQ ID NOS: 27-38.

34. The array of claim 31 , wherein the at least three isolated nucleic acid molecules consist of SEQ ID NOS: 27-41 and 43.

35. The array of claim 31, wherein the at least three isolated nucleic acid molecules consist of SEQ ID NOS: 27-43.

36. The array of claim 31, wherein the isolated nucleic acid molecules are chemically linked to the solid support surface in a predetermined pattern.

37. The array of claim 36, wherein the solid support is a polymeric solid support.

38. A kit for detecting HBV, HCV, and HIV-I in a sample, comprising: the array of claim 31 ; and a buffer solution, in separate packaging.

39. The kit of claim 38, further comprising primers capable of amplification of an S-gene HBV sequence, primers capable of amplification of a 5'-UTR HCV sequence, and primers capable of amplification of a gp41 HIV-I sequence.

40. The kit of claim 39, wherein the primers capable of amplification of an S-gene HBV sequence comprise SEQ ID NOS: 20-23, the primers capable of amplification of a 5'-UTR HCV sequence comprise SEQ ID NOS: 16-19, and the primers capable of amplification of a gp41 HIV-I sequence comprise SEQ ID NOS: 12-15.

41. The kit of claim 40, wherein the primers capable of amplification of an S-gene HBV sequence consist of SEQ ID NOS: 20-23, the primers capable of amplification of a 5'-UTR HCV sequence consist of SEQ ID NOS: 16-19, and the primers capable of amplification of a gp41 HIV-I sequence consist of SEQ ID NOS: 12-15.