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  Assay for detecting and quantifying hiv-1USPTO Application #: 20060068380Title: Assay for detecting and quantifying hiv-1 Abstract: Compositions, methods and kits for detecting HIV-1 nucleic acids using nucleic acid amplification. Particularly described are oligonucleotides that are useful as hybridization probes and amplification primers for detecting very low levels of HIV-1 nucleic acids by real-time monitoring of amplicon production. The invented assays are characterized by high levels of precision in the quantitation of HIV-1 targets at low copy numbers, and by accurate detection of different HIV-1 subtypes, including M group and O group variants. (end of abstract) Agent: Gen Probe Incorporated - San Diego, CA, US Inventors: Astrid R.W. Schroder, Glenn J. Sawyer, Daniel P. Kolk USPTO Applicaton #: 20060068380 - Class: 435005000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Virus Or Bacteriophage The Patent Description & Claims data below is from USPTO Patent Application 20060068380. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application Nos. 60/615,533, filed Sep. 30, 2004. The entire disclosure of this prior application is hereby incorporated by reference. FIELD OF THE INVENTION [0002] The present invention relates to the field of biotechnology. More specifically, the invention relates to diagnostic assays for detecting and quantifying the nucleic acids of HIV-1. BACKGROUND OF THE INVENTION [0003] Advances in the clinical management of individuals infected with the human immunodeficiency virus type 1 (HIV-1) have been able to reduce viral titers below the detection limits of some early-generation HIV-1 assays. More specifically, highly active anti-retroviral drug therapy (HAART) can reduce the viral load down to a level approaching 50 HIV-1 RNA copies/ml, a level substantially below the 400-500 copies/ml threshold of some previous detection assays. This fact, together with a desire to monitor and maintain low viral titers, necessitated the development of improved quantitative assays for measuring HIV-1 RNA. (Elbeik et al., J. Clin. Micro. 38:1113-1120 (2000)) Complicating matters, however, is the fact that useful quantitative assays must be capable of accurately measuring a range of genetically diverse HIV-1 variants. [0004] Three classes of HIV-1 have developed across the globe: M (major), O (outlying) and N (new). Among the M group, which accounts for greater than 90% of reported HIV/AIDS cases, viral envelopes have diversified so greatly that this group has been subclassified into nine major clades including A-D, F-H, J and K, as well as several circulating recombinant forms. Subtypes within the HIV-1 O group are not clearly defined, and the diversity of sequences within the O group is nearly as great as the diversity of sequences in the HIV-1 M group. Phylogenetic analyses of the gag and env genes have failed to reveal clades of O group viruses as clearly as the clades detected in the M group. Subtypes and sub-subtypes of the HIV-1 M group are thought to have diverged in humans following a single chimpanzee-to-human transmission event. In contrast, the HIV-1 O and N groups are each thought to have resulted from separate chimpanzee-to-human transmission events. Of the completely sequenced HIV-1 genomes, nearly 20% have a mosaic structure consisting of at least two subtypes, yet another potential complication for ultrasensitive HIV-1 detection assays. (Spira et al., J. Antimicrobial Chemotherapy 51:229 (2003).) [0005] Most viral load monitoring is currently performed in the developed Western World where the clade B (i.e., "subtype B" hereafter), which represents only about 3% of HIV infections worldwide, predominates. Importantly, the HIV-1 viral subtypes are expanding in different geographical regions, thereby imposing an additional requirement for broad detection capacity on detection and viral load monitoring assays. Accordingly, there is a need for ultrasensitive HIV-1 detection assays which are capable of accurately measuring the full range of HIV-1 subtypes. The present invention addresses this need. [0006] An example quantitative HIV-1 assay, performed using real-time monitoring of a nucleic acid amplification reaction, has been described in published International Patent Application WO 2003106714. SUMMARY OF THE INVENTION [0007] A first aspect of the invention relates to a reaction mixture useful for amplifying either HIV-1 M group nucleic acids or HIV-1 O group nucleic acids. The invented reaction mixture ordinarily includes first and second amplification primers. The first amplification primer includes a first primer target-hybridizing sequence that can independently hybridize to a first strand of HIV-1 M group nucleic acids, and to a first strand of HIV-1 O group nucleic acids. The second amplification primer includes a second primer target-hybridizing sequence that hybridizes to an enzymatic extension product of the first amplification primer using as a template either the first strand of HIV-1 M group nucleic acids or the first strand of HIV-1 O group nucleic acids. The second primer target-hybridizing sequence consists essentially of SEQ ID NO:33. In a preferred embodiment, the second primer target-hybridizing sequence consists essentially of SEQ ID NO:2. When this is the case, the first primer target-hybridizing sequence may consist essentially of SEQ ID NO:13. Alternatively, the first primer target-hybridizing sequence may consist essentially of SEQ ID NO:15. In a different preferred embodiment, the second primer target-hybridizing sequence consists essentially of SEQ ID NO:5. When this is the case, the first primer target-hybridizing sequence may consist essentially of SEQ ID NO:15. In yet another preferred embodiment, the reaction mixture further includes a hybridization probe. In some instances, the hybridization probe is a molecular beacon hybridization probe or a molecular torch hybridization probe. Regardless of whether the hybridization probe is a molecular beacon or a molecular torch, it is preferred in certain embodiments that no more than two primers and a single probe are used for amplifying and detecting the HIV-1 M group nucleic acids or the HIV-1 O group nucleic acids. [0008] A second aspect of the invention relates to a method of quantifying the combined amount of an HIV-1 M group nucleic acid and an HIV-1 O group nucleic acid that may be present in a biological sample. The invented method involves steps for: (a) combining in a single reaction vessel the biological sample, a first amplification primer, a second amplification primer, and a hybridization probe; (b) amplifying, with substantially equal efficiency, any of the HIV-1 M group nucleic acid and the HIV-1 O group nucleic acid present in the biological sample using an in vitro amplification reaction that relies on enzymatic extension of the first amplification primer using a first strand of the HIV-1 M group nucleic acid or the HIV-1 O group nucleic acid as a first template to create a first primer extension product, and enzymatic extension of the second amplification primer using the first primer extension product as a second template, whereby there are produced HIV-1 M group amplicons if the biological sample contained HIV-1 M group nucleic acids, and HIV-1 O group amplicons if the biological sample contained HIV-1 O group nucleic acids; (c) monitoring amplicon production in the in vitro amplification reaction as a function of time by a process that includes detection of a signal from the hybridization probe, whereby time-dependent quantitative data is obtained; and (d) quantifying the combined amount of the HIV-1 M group nucleic acid and the HIV-1 O group nucleic acid present in the biological sample using the time-dependent quantitative data obtained in the monitoring step. In accordance with this aspect of the invention, neither the first amplification primer nor the second amplification primer is fully complementary to the HIV-1 M group nucleic acid or the complement thereof, or to the HIV-1 O group nucleic acid or the complement thereof. Further in accordance with this aspect of the invention, the hybridization probe hybridizes to both HIV-1 M group amplicons and HIV-1 O group amplicons. Notably, the invented method also is contemplated for use in detecting and quantifying HIV-1 N group nucleic acids. In a preferred embodiment, the in vitro amplification reaction is an isothermal in vitro amplification reaction that does not require temperature cycling to achieve some degree of exponential amplification. More preferably, the isothermal in vitro amplification reaction is a transcription associated amplification reaction that is either a TMA reaction or a NASBA reaction. In an alternative preferred embodiment, the signal detected in the monitoring step is a fluorescent signal, such as a fluorescent signal produced by a molecular torch hybridization probe. In a highly preferred embodiment, the first amplification primer includes a first primer target-hybridizing sequence that consists essentially of SEQ ID NO:15. More preferably, the second amplification primer includes a second primer target-hybridizing sequence that consists essentially of SEQ ID NO:5. In accordance with another preferred embodiment, no more than two primers and a single probe are used for amplifying and detecting both the HIV-1 M group nucleic acid and the HIV-1 O group nucleic acid. In a highly preferred embodiment, the in vitro amplification reaction is an isothermal in vitro amplification reaction. In an alternative highly preferred embodiment, the quantifying step involves comparing a quantitative result with no more than a single standard curve. [0009] A third aspect of the invention relates to a method of establishing a point on a standard curve that can be used for quantifying HIV-1 M group nucleic acids and HIV-1 O group nucleic acids in a single reaction. The invented method involves steps for: (a) providing a known amount of an HIV-1 standard; (b) amplifying in an in vitro amplification reaction the HIV-1 standard using a first primer and a second primer in the presence of a hybridization probe to produce HIV-1 standard amplicons, wherein the amplification reaction amplifies HIV-1 M group nucleic acids and HIV-1 O group nucleic acids with substantially equal efficiency; (c) monitoring production of HIV-1 standard amplicons synthesized in the in vitro amplification reaction as a function of time by a process that involves detection of a signal from the hybridization probe, whereby quantitative data is obtained; and (d) establishing from the quantitative data a point on the standard curve. In a preferred embodiment, the first amplification primer includes a first primer target-hybridizing sequence that independently hybridizes to a first strand of HIV-1 M group nucleic acids and to a first strand of HIV-1 O group nucleic acids, wherein the second amplification primer includes a second primer target-hybridizing sequence that hybridizes to an enzymatic extension product of the first amplification primer using as a template either the first strand of HIV-1 M group nucleic acids or the first strand of HIV-1 O group nucleic acids. In accordance with this embodiment, (a) neither the first primer target-hybridizing sequence nor the second primer target-hybridizing sequence is fully complementary to HIV-1 M group or HIV-1 O group nucleic acids or the complements thereof, and (b) the hybridization probe is able to hybridize either to HIV-1 M group nucleic acids and HIV-1 O group nucleic acids, or to their complements. In one preferred embodiment, the hybridization probe is a molecular torch. More preferably, when the hybridization probe is a molecular torch, the HIV-1 standard is an HIV-1 M group nucleic acid standard. Still more preferably, when the hybridization probe is a molecular torch, and when the HIV-1 standard is an HIV-1 M group nucleic acid standard, there can be an additional step for using the standard curve to quantify an HIV-1 O group nucleic acid contained in a biological sample. In a different preferred embodiment, the HIV-1 standard is an HIV-1 O group nucleic acid standard. When this is the case, there can be a further step for using the standard curve to quantify an HIV-1 M group nucleic acid contained in a biological sample. In still another different embodiment, the in vitro amplification reaction in the amplifying step can be an isothermal in vitro amplification reaction. When this is the case, the isothermal in vitro amplification reaction can be a transcription associated amplification reaction, such as a TMA reaction or a NASBA reaction. In such an instance, the step for monitoring can involve measuring a fluorescent signal. [0010] A fourth aspect of the invention relates to a method of preparing a reaction mixture for amplifying either or both of HIV-1 M group nucleic acids and HIV-1 O group nucleic acids. The invented method includes steps for: (a) selecting a first amplification primer that includes a sequence that independently hybridizes to a first strand of either HIV-1 M group target nucleic acids or HIV-1 O group target nucleic acids; (b) selecting a second amplification primer that includes a sequence that hybridizes to enzymatic extension products of the first amplification primer using the first strand of either HIV-1 M group target nucleic acids or HIV-1 O group target nucleic acids as a template; (c) selecting a hybridization probe that hybridizes to amplicons synthesized by the use of the first and the second amplification primers, wherein neither the first primer target-hybridizing sequence nor the second primer target-hybridizing sequence is fully complementary to the HIV-1 M group or HIV-1 O group nucleic acids or the complements thereof, and wherein the first amplification primer, the second amplification primer, and the hybridization probe are further selected to amplify in an in vitro amplification reaction HIV-1 M group nucleic acids and HIV-1 O group nucleic acids with substantially equal efficiencies; and (d) combining in a single reaction vessel the first amplification primer, the second amplification primer, and the hybridization probe. In a preferred embodiment, the reaction mixture includes no more than two primers and a single hybridization probe for amplifying and detecting the HIV-1 M group nucleic acids and HIV-1 O group nucleic acids. More preferably, the in vitro amplification reaction is an isothermal in vitro amplification reaction. Still more preferably, the isothermal in vitro amplification reaction is a transcription associated amplification reaction that is either a TMA reaction or a NASBA reaction. [0011] A fifth aspect of the invention relates to a composition for amplifying HIV-1 M group target nucleic acids and HIV-1 O group target nucleic acids. The invented composition includes: (a) a first amplification primer that includes a first primer target-hybridizing sequence that independently hybridizes to a first strand of HIV-1 M group target nucleic acids and to a first strand of HIV-1 O group target nucleic acids; and (b) a second amplification primer that includes a second primer target-hybridizing sequence that hybridizes to enzymatic extension products of the first amplification primer using the first strand of either HIV-1 M group target nucleic acids or HIV-1 O group target nucleic acids as a template. In accordance with this aspect of the invention, neither the first primer target-hybridizing sequence nor the second primer target-hybridizing sequence is fully complementary to the HIV-1 M group or HIV-1 O group target nucleic acids or the complements thereof. In a preferred embodiment, the composition also includes a hybridization probe that hybridizes to an amplification product produced in an in vitro amplification reaction by the combined activity of the first and second amplification primers using as a template either HIV-1 M group target nucleic acids or HIV-1 O group target nucleic acids. More preferably, the composition amplifies HIV-1 M group target nucleic acids and HIV-1 O group target nucleic acids in the in vitro nucleic acid amplification reaction with substantially equal efficiency. Still more preferably, the first primer target-hybridizing sequence consists essentially of SEQ ID NO:15, and the second primer target-hybridizing sequence consists essentially of SEQ ID NO:5. In an alternative preferred embodiment, the hybridization probe is a molecular torch or a molecular beacon. In certain instances, it is preferred for the hybridization probe to be a molecular torch. In other preferred embodiments, the second primer target-hybridizing sequence consists essentially of SEQ ID NO:5. When this is the case, the first primer target-hybridizing sequence may consist essentially of SEQ ID NO:15. In still other preferred embodiments, when the composition includes the above-mentioned hybridization probe, the first primer target-hybridizing sequence may consist essentially of SEQ ID NO:15, and the second primer target-hybridizing sequence may consist essentially of SEQ ID NO:5. More preferably, the hybridization probe is a molecular torch. [0012] A sixth aspect of the invention relates to a reaction mixture for amplifying either HIV-1 M group nucleic acids or HIV-1 O group nucleic acids. The invented reaction mixture includes: (a) a first amplification primer that includes a first primer target-hybridizing sequence that independently hybridizes to a first strand of HIV-1 M group nucleic acids and a first strand of HIV-1 O group nucleic acids; (b) a second amplification primer that includes a second primer target-hybridizing sequence that hybridizes to an enzymatic extension product of the first amplification primer, using as a template either the first strand of HIV-1 M group nucleic acids or the first strand of HIV-1 O group nucleic acids; and (c) a molecular torch hybridization probe that hybridizes to an amplicon synthesized by the combined activity of the first amplification primer and the second amplification primer. In accordance with this aspect of the invention, neither the first primer target-hybridizing sequence nor the second primer target-hybridizing sequence is fully complementary to the HIV-1 M group or HIV-1 O group nucleic acids or the complements thereof. Significantly, the HIV-1 M group nucleic acids and HIV-1 O group nucleic acids amplify in the reaction mixture with substantially equal efficiency. DEFINITIONS [0013] The following terms have the following meanings for the purpose of this disclosure, unless expressly stated to the contrary herein. [0014] As used herein, a "biological sample" is any tissue or polynucleotide-containing material obtained from a human, animal or environmental sample. Biological samples in accordance with the invention include peripheral blood, plasma, serum or other body fluid, bone marrow or other organ, biopsy tissues or other materials of biological origin. A biological sample may be treated to disrupt tissue or cell structure, thereby releasing intracellular components into a solution which may contain enzymes, buffers, salts, detergents and the like. [0015] As used herein, "polynucleotide" means either RNA or DNA, along with any synthetic nucleotide analogs or other molecules that may be present in the sequence and that do not prevent hybridization of the polynucleotide with a second molecule having a complementary sequence. [0016] As used herein, a "detectable label" is a chemical species that can be detected or can lead to a detectable response. Detectable labels in accordance with the invention can be linked to polynucleotide probes either directly or indirectly, and include radioisotopes, enzymes, haptens, chromophores such as dyes or particles that impart a detectable color (e.g., latex beads or metal particles), luminescent compounds (e.g., bioluminescent, phosphorescent or chemiluminescent moieties) and fluorescent compounds. [0017] A "homogeneous detectable label" refers to a label that can be detected in a homogeneous fashion by determining whether the label is on a probe hybridized to a target sequence. That is, homogeneous detectable labels can be detected without physically removing hybridized from unhybridized forms of the label or labeled probe. Homogeneous detectable labels are preferred when using labeled probes for detecting HIV-1 nucleic acids. Examples of homogeneous labels have been described in detail by Arnold et al., U.S. Pat. No. 5,283,174; Woodhead et al., U.S. Pat. No. 5,656,207; and Nelson et al., U.S. Pat. No. 5,658,737. Preferred labels for use in homogenous assays include chemiluminescent compounds (e.g., see Woodhead et al., U.S. Pat. No. 5,656,207; Nelson et al., U.S. Pat. No. 5,658,737; and Arnold, Jr., et al., U.S. Pat. No. 5,639,604). Preferred chemiluminescent labels are acridinium ester ("AE") compounds, such as standard AE or derivatives thereof (e.g., naphthyl-AE, ortho-AE, 1- or 3-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE, 1- or 3-methyl-meta-difluoro-AE, and 2-methyl-AE). [0018] A "homogeneous assay" refers to a detection procedure that does not require physical separation of hybridized probe from non-hybridized probe prior to determining the extent of specific probe hybridization. Exemplary homogeneous assays, such as those described herein, can employ molecular beacons or other self-reporting probes which emit fluorescent signals when hybridized to an appropriate target, chemiluminescent acridinium ester labels which can be selectively destroyed by chemical means unless present in a hybrid duplex, and other homogeneously detectable labels that will be familiar to those having an ordinary level of skill in the art. [0019] As used herein, "amplification" or "amplifying" refers to an in vitro procedure for obtaining multiple copies of a target nucleic acid sequence, its complement or fragments thereof. Continue reading... Full patent description for Assay for detecting and quantifying hiv-1 Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Assay for detecting and quantifying hiv-1 patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. 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