WO2023126629A1

WO2023126629A1 - Method for detecting and/or quantifying a virus

Info

Publication number: WO2023126629A1
Application number: PCT/GB2022/053401
Authority: WO
Inventors: Catherine KIBIRIGE; Mark Manak; Linda JAGODZINSKI
Original assignee: Imperial College Innovations Limited; The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc.; The Government Of The United States, As Represented By The Secretary Of The Army
Priority date: 2021-12-31
Filing date: 2022-12-30
Publication date: 2023-07-06

Abstract

The present invention relates to methods for detecting and/or quantifying a virus, particularly at low concentrations. In particular, the invention relates to the detection and/or quantification of HIV-1 using different regions of the Long Term Repeats (LTR) as targets for the amplification. The invention also relates to oligonucleotides useful in said methods, particularly primer and probe nucleic acids, as well as kits suitable for carrying out said methods.

Description

METHOD FOR DETECTING AND/OR QUANTIFYING A VIRUS

This invention was made with U.S. Government support under AID0AA-A-16-00032. The U.S. Government has certain rights in this invention.

This work has been supported in whole or part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Contract Number NOI-AI-85341.

This invention was made with government support under W81XWH-11-2-0174 awarded by the United States Army Medical Research and Development Command. The government has certain rights in the invention.

This invention was made with the support of the World Health Organization and the United Nations Programme on HIV/AIDS, as well as the National Institute of Health AIDS Research and Reference Reagent Program.

FIELD OF THE INVENTION

The present invention relates to methods for detecting and/or quantifying a virus, particularly at low concentrations. In particular, the invention relates to the detection and/or quantification of HIV-1. The invention also relates to oligonucleotides useful in said methods, particularly primer and probe nucleic acids, as well as kits suitable for carrying out said methods.

BACKGROUND OF THE INVENTION

In order to effectively monitor viral therapies and support cures studies, there is a need for relatively simple, low-cost sensitive assays that can reliably quantify low levels of a given virus, including across different strains. This is particularly the case for HIV-1 combination anti-retroviral therapy (cART) and HIV-1 cure studies. HIV suppression, following initiation of cART, to levels that are undetectable by commercial viral load assays (i.e., below 20 to 50 copies/ml in plasma), is significantly associated with longevity and improved quality of life. Moreover, since successfully treated people do not transmit HIV-1, early and sustained viral suppression is an important public health tool in the prevention of the spread of HIV. Current WHO guidelines thus strongly recommend initiation of cART as soon as possible after HIV-1 diagnosis.

Interestingly, the use of highly sensitive laboratory developed assays (LDAs), such as the Single-Copy Assay (SCA) which detects down to 1 copy of HIV-1 RNA per ml of plasma, has shown that discontinuation of antiretroviral treatment by HIV infected patients with plasma RNA levels above 10 - 15 copies / ml results in viremia rebound earlier and more often than patients who have lower or undetectable RNA. These results indicate that candidates for viral control or cure studies may have better success if viral loads are driven below 15 copies/ml before treatment cessation.

Integrating viruses, such as HIV-1 integrate their DNA into the human chromosome and persist in an inactive state even in the presence of ongoing antiretroviral therapy. This means that reduction of plasma viral load to low levels, even below 10 - 15 copies / ml as indicated by the highly efficient SCA assay, does not lead to viral eradication. The integrated viral DNA serves as a long-term reservoir for viral reinfection and can be reactivated when therapy is discontinued or when drug resistant isolates emerge. Despite dramatic decreases in plasma HIV-1 RNA levels following treatment with cART, levels of integrated viral DNA in virally suppressed subjects persist at similar levels to those of untreated subjects. HIV-1 viral reservoir sites include CD4 memory T cells, lymph nodes, the gut and other tissues. To measure the levels of replication-competent integrated DNA within these reservoir sites, various assays have been developed. The quantitative virus outgrowth assay (Q.VOA) is considered the gold-standard assay for this type of assessment, but is complicated, expensive and time intensive, and despite recent improvements, is not practical for routine viral monitoring.

Alternative assays have been developed to quantify cell-associated viral nucleic acids for measurement of residual quiescent viral genomes. Indeed, several studies have shown that viral nucleic acids can be readily detected in whole blood and peripheral blood mononuclear cells (PBMCs) of HIV-1 infected individuals even when plasma viral load is very low or undetectable. The level of residual HIV-1 DNA in the PBMCs of treated subjects ranges from 100 to 10,000 copies of HIV-1 DNA per million cells and remains sufficiently high as to allow for reliable detection of quiescent infection, with only those who have initiated therapy during Fiebig disease stage I or II at lower levels. These alternative cell-based PCR assays have been shown to correlate well with the Q.VOA and thus the latent viral reservoir. These simplified assays are used as a proxy for viral reservoir estimation and provide a more efficient tool for large-scale patient screening and research because they can be tailored to the available specimens and can be run at lower cost (~<$4 per test) compared to FDA-approved plasma viral load assays ($50 - 100+ per test).

However, whilst offering some practical improvements over Q.VOA, a significant problem remaining with these alternative assays is their sensitivity and specificity against different viral subtypes. Many of the LDAs for HIV-1 quantification used in clinical research were developed against HIV-1 subtype B, the predominant subtype circulating in Europe and America. This subtype only accounts for 10% of the infections worldwide. Rapid spread of HIV in Africa and Asia and increased international travel have led to a steady increase in non-B HIV-1 in the western hemisphere. Current HIV-1 isolates have been classified into three major groups (M, N and O) and further subdivided into subtypes or clades, various circulating recombinant forms (CRFs) and unique recombinant forms (URFs) spanning up to 40% sequence divergence. Therefore, a major challenge in the design and implementation of LDAs for HIV-1, is the large worldwide sequence diversity of the virus and the lack of ability of current LDAs to cover the broad range of HIV-1 subtypes in various target populations. Moreover, the extensive variability in sample processing and amplification procedures among the various LDAs, makes it difficult to compare subtype sensitivity and specificity between different test formats and laboratories.

Accordingly, there is an unmet need to provide LDAs that are able to quantify and/or detect viruses with high sensitivity and specificity, and to do so at low viral concentrations and across different viral subtypes, particularly for HIV-1. It is an object of the present invention to address at least one of these problems. In particular, the present invention aims to reduce the cost and provide a method for viral detection and/or quantification that is sensitive, specific and reliable across different viral subtypes, and enables detection and/or quantification of virus from small amounts of a range of sample types. Such a method will facilitate monitoring for viral therapies and support cure studies, particularly for HIV-1.

SUMMARY OF THE INVENTION

The present inventors are the first to develop a sensitive polymerase chain reaction (PCR)- based assay which can be used in various formats to detect residual viral nucleic acids in plasma and PBMC of infected individuals undergoing therapy. In particular, the inventors have developed an assay that can detect residual HIV-1 nucleic acids in plasma and PBMC of HIV-1 infected individuals undergoing cART. In a liquid format, the assay can detect 88 copies of HIV-1 RNA per ml with 95% efficiency. The semi-nested oligonucleotide sets allow for further sensitivity of the liquid format in laboratories where carry-over contamination can be circumvented. In the alternative cellular format, the assay can detect down to 3 input copies of nucleic acid or the equivalent of a single infected cell at the 95% confidence level even in a non-nested format. Compared to previously reported laboratory developed assays (LDAs), the invention assay provides improved efficiency, a very broad dynamic range and superior cross-subtype specificity. Further, the inventors have evaluated the methods of the invention assay according to minimum information required for the publication of quantitative real-time PCR experiments (MIQ.E) guidelines using an extensive diversity panel of HIV-1 strains provided by the NIH/NIAID/DIAIDS External Quality Assurance Program Oversight Laboratory (EQAPOL) and the United States Military HIV Research Program (US MHRP) via the AIDS Reference Reagent Program. The inventors have demonstrated that the methods of the invention have at least comparable sensitivity to current commercial PCR-based HIV-1 assays used for plasma viral load testing (in the non-nested format) and for sensitive detection of cell-associated HIV-1 levels in PBMC but are considerably cheaper to perform which greatly increases their utility in resource-limited settings.

Accordingly the invention provides an in vitro method of detecting and/or quantifying a virus, which method comprises or consists of the steps of: (a) identifying an oligonucleotide region that is conserved across multiple different strains of a virus by aligning a diversity panel of a plurality of different strains of said virus; (b) designing one or more primer sequence that is suitable for amplification of the identified nucleotide region, wherein said one or more primer sequence is specific to the identified nucleotide region, wherein any mismatches between the sequences of the plurality of different strains identified by the alignment are corrected; and (c) detecting and/or quantifying the virus within a biological sample by amplifying viral nucleic acid using the one or more primer sequence; wherein optionally the diversity panel comprises sequences from at least 20 strains of said virus.

The invention also provides an in vitro method of detecting and/or quantifying a virus, which method comprises the steps of: (a) amplifying viral nucleic acid in a biological sample with PCR using a first and second primer for a viral LTR sequence, wherein the first and second primers hybridise to different regions within the LTR sequence, and wherein said first primer is linked to a tag sequence; (b) subjecting the amplified nucleic acid from step (a) to another amplification with PCR using the second primer and a primer for the tag sequence; and (c) detecting and/or quantifying the nucleic acid that was amplified through steps (a) and (b), wherein the detected/quantified nucleic acid correlates with the number of copies of the virus genome.

Said first and/or second primer may comprise at least one inosine base. Said first and second primers may hybridise to different regions of the 5' LTR sequence. The first primer may hybridise to the R region of the 5' LTR sequence; and/or the second primer may hybridise to the U5 region of the 5' LTR sequence. The first primer may comprise or consist of SEQ ID NO: 1, or a variant differing by one or two nucleotides from SEQ. ID NO: 1; and/or the second primer may comprise or consist of SEQ ID NO: 2, or a variant differing by one or two nucleotides from SEQ ID NO: 2.

The method of the invention may be used to detect and/or quantify total viral RNA and/or DNA.

The invention further provides an in vitro method of detecting and/or quantifying an integrated virus, which method comprises the steps of: (a) amplifying viral nucleic acid in a biological sample with PCR using a primer for an Alu sequence and a first primer for a viral LTR sequence, wherein said first primer for a viral LTR sequence primer is linked to a tag sequence; (b) subjecting the amplified nucleic acid from step (a) to another amplification with PCR using a primer for the tag sequence and a second primer for the viral LTR sequence; and (c) detecting and/or quantifying the nucleic acid that was amplified through steps (a) and (b), wherein the detected/quantified nucleic acid correlates with the number of copies of the integrated virus genome. In step (b) the primer for the tag sequence is typically used as the forward primer. The second primer for the viral LTR sequence hybridises to a different region of the viral LTR sequence than the first LTR primer used in step (a). Typically the second primer for the viral LTR sequence hybridises to a region upstream of the target region for the first LTR primer, and as such is used as the reverse primer in step (b).

Said Alu sequence primer and/or viral LTR primers may comprise at least one inosine base. The first and/or second primer for the viral LTR sequence typically hybridise to different regions of the viral LTR sequence. The first primer may hybridise to the R region and/or the second primer may hybridise to the U5 region of the 5' LTR sequence. The first primer for the viral LTR sequence in step (a) may comprise or consist of the nucleic acid selected from SEQ ID NO: 1 (or a variant differing by one or two nucleic acids from SEQ. ID NO: 1) and SEQ ID NO 3 (or a variant differing by one or two nucleotides from SEQ ID NO: 3). The second primer for the viral LTR sequence in step (b) may comprise or consist of a nucleic acid selected from SEQ ID NO: 2 (or a variant differing by one or two nucleotides from SEQ ID NO: 2).

The virus may be a retrovirus or lentivirus, wherein optionally the retrovirus is HIV, preferably HIV-1.

The tag sequence or the primer for the tag sequence: (a) may be a sequence not found in the virus or host organism; and/or (b) may comprise or consist of a bacteriophage lambda nucleotide sequence, optionally SEQ ID NO: 4, or a variant differing by one or two nucleotides from SEQ ID NO: 4.

For PCR-based methods of the invention, the PCR of step (b) may be performed in the presence of at least one detectable probe that specifically hybridises with the viral nucleic acid amplified, wherein the hybridisation of the probe allows for the detection and/or quantification of the virus.

The probe may: (a) comprise a fluorescent moiety; (b) comprise a 5' FAM, VIC, TET or NED dye, and optionally a 3' non-fluorescent quencher (NFQ), wherein preferably said probe further comprises a 3' minor groove binder moiety (MGB); and/or (c) comprise or consist of SEQ ID NO: 5, or a variant differing by one or two nucleotides from SEQ ID NO: 5, and preferably wherein said probe is 5'6- F AM/ACAG AYGGGC AC AC AC I ACT/M G B N FQ-3' .

A method of the invention may be used with a sample which comprises or consists of fluid and/or cells from a subject, preferably wherein said sample comprises or consists of: (a) plasma; (b) peripheral blood mononuclear cells (PBMCs); and/or (c) PBMC lysate.

For PCR-based methods of the invention: (a) step (a) may comprise or consist of from 10 to 15 cycles of PCR; and/or (b) step (b) may comprise or consist of from 30 to 50 cycles of PCR.

For PCR-based methods of the invention: (a) the PCR cycles may comprise or consist of step-up PCR cycles; (b) the PCR may be qPCR or RTqPCR; (c) a ten-fold dilution may be carried out between steps (a) and (b); (d) each PCR reaction may comprise tRNA, optionally at a concentration of 10 ng/mL; and/or (e) the lower limit of detection of the target organism may be (i) about 80 to 90 copies/mL, or (ii) about 3 input copies of nucleic acid.

The methods of the invention may use one or more primer or probe sequence designed using a method of identifying and designing a primer sequence by a method comprising or consisting of the steps of (a) identifying an oligonucleotide region that is conserved across multiple different strains of a virus by aligning a diversity panel of a plurality of different strains of said virus; and (b) designing one or more primer sequence that is suitable for amplification of the identified nucleotide region, wherein said one or more primer sequence is specific to the identified nucleotide region, wherein any mismatches between the sequences of the plurality of different strains identified by the alignment are corrected.

A method of the invention may be a multiplex method in which one or more additional target region of the viral genome is detected and/or quantified, wherein optionally said one or more additional target region is selected from a viral integrase gene and/or a viral polymerase gene.

The invention also provides an oligonucleotide primer which comprises or consists of any one of SEQ ID NOs: 1 to 4 or a variant differing by one or two nucleotides from any one of SEQ ID NOs: 1 to 4.

The invention further provides an oligonucleotide probe which comprises or consists of SEQ. ID NO: 5, or a variant differing by one or two nucleotides from SEQ ID NO: 5.

The invention further provides a set of oligonucleotides comprising or consisting of: (a) (i) SEQ ID NO 3, or a variant differing by one or two nucleotides from SEQ ID NO: 3; or (ii) SEQ ID NO: 1, or a variant differing by one or two nucleotides from SEQ ID NO: 1 and SEQ ID NO: 4, or a variant differing by one or two nucleotides from SEQ ID NO: 4; (b) SEQ ID NO 2, or a variant differing by one or two nucleotides from SEQ ID NO: 2; and (c) SEQ ID NO: 5, preferably 5'6- FAM/ACAGAYGGGCACACACIACT/MGBNFQ-3', or a variant differing by one or two nucleotides from SEQ ID NO: 5.

The invention also provides a kit for performing a method of the invention, which comprises or consists of: (a) (i) a first container containing a first and second primer for a viral LTR sequence and a second container containing the second primer for a viral LTR sequence and a primer for the tag sequence; or (ii)a first container containing a first primer for a viral LTR sequence, a second container containing a second primer for a viral LTR sequence, and a third container containing a primer for the tag sequence; and (iii) a detectable probe, wherein the detectable probe may be contained in same container as the second primer and/or tag primer, or in a separate container; or (b) a first container containing an Alu primer and a first primer for a viral LTR sequence and a second container containing a second primer for a viral LTR sequence and a primer for the tag sequence; or (ii) a first container containing an Alu primer, a second container containing a first primer for a viral LTR sequence, a third container containing a second primer for a viral LTR sequence, and a fourth container containing a primer for the tag sequence; and (iii) a detectable probe, wherein the detectable probe may be contained in same container as the second primer for a viral LTR sequence and/or tag primer, or in a separate container; wherein optionally said kit may comprise one or more additional reagent for carrying out said method.

The invention further provides an in vitro method of detecting and/or quantifying a virus, comprising carrying out PCR using one or more oligonucleotides primers of the invention and/or one or more oligonucleotide probe of the invention, wherein preferably said method comprises carrying out PCR using a set of oligonucleotides of the invention, and wherein optionally said PCR is: (a) qPCR or RT-qPCR; and/or (b) nested or semi-nested PCR.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Different PCR-based method formats/protocols of the invention and recommended uses

Figure 2: Revised qPCR and RTqPCR quantification assay synthetic standard and formats, a. Synthetic DNA plasmid template containing the 433-633 HIV-1 LTR insert was linearized by Seal, and dilutions used as a standard curve in the HIV-1 LDAs. HIV-1 specific transcripts from the linearized plasmid were made using the T7 promoter site for RNA transcription and were used to make HIV RNA controls, b. Exemplary protocol for Non-nested qPCR on crude DNA lysates, c. Exemplary protocol for semi-nested RT-qPCR on RNA extracts.

Figure 3: Optimization and Validation of Modified LTR-based qPCR and RTqPCR Assay Formats, a. Optimization of cycling condition of the initial 496F/546P/633R Brussel primer-probe set on total HIV- 1 DNA detection using universal cycling with tRNA; step-up cycling with no tRNA or step-up cycling with 10 ng/mL tRNA. Geometric means and standard errors of means are plotted for 3 to 12 replicates per sample, b. Evaluation of the linearity of the RT-qPCR format of the 525F/574P/599R revised assay for detection of HIV-1 RNA on an AcroMetrix HIV-1 Linearity Panel, c. Demonstration of the linearity qPCR format of the revised laboratory developed assay for detection of total HIV-1 nucleic acids on dilutions of crude lysates of 8E5 cells, d. Plasma viral load limit of detection (LOD) determined of revised laboratory developed assay by probit regression of serial dilutions of EDTA plasma spiked with a HIV-1 AcroMetrix quantification standard near the cut-off level of the assay. The percentage of specimens detected at each copy level is indicated, and the 50% and 95 % detection level extrapolated from the curve. Three replicates were used for 100 copies/ml while 10 replicates were used for the other dilutions, e. Correlation in RNA plasma viral load measurements obtained by the revised laboratory developed assay and the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test v2.0 assay. The analysis was performed on 127 high-titer HIV-1 spiked plasmas from the External Quality Assurance Program Oversite Laboratory (EQAPOL) and the US Military's HIV Research Program (USMHRP). f. Bland-Altman plot showing the difference between the measurements obtained by the revised laboratory developed assay and the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test v2.0 assay. The analysis was performed on 127 high-titer HIV-1 spiked plasmas from the External Quality Assurance Program Oversite Laboratory (EQAPOL) and the US Military's HIV Research Program (USMHRP). g. A typical set of assay amplification curves obtained using the revised laboratory developed assay, h. A typical standard curve obtained using the revised laboratory developed assay

Figure 4: Comparison of HIV-1 viral load measurements by HIV-1 subtype determined by the revised laboratory developed assay (LDA) compared to the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test v2.0 assay.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.

As used herein, the term "capable of' when used with a verb, encompasses or means the action of the corresponding verb. For example, "capable of interacting" also means interacting, "capable of cleaving" also means cleaves, "capable of binding" also means binds and "capable of specifically targeting..." also means specifically targets.

Numeric ranges are inclusive of the numbers defining the range. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

As used herein, the articles "a" and "an" may refer to one or to more than one (e.g. to at least one) of the grammatical object of the article. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting.

"About" may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. Preferably, the term "about" shall be understood herein as plus or minus (±) 5%, preferably ± 4%, ± 3%, ± 2%, ± 1%, ± 0.5%, ± 0.1%, of the numerical value of the number with which it is being used.

The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the invention.

As used herein the term "consisting essentially of" refers to those elements required for a given invention. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that invention (i.e. inactive or non-immunogenic ingredients).

Embodiments described herein as "comprising" one or more features may also be considered as disclosure of the corresponding embodiments "consisting of" and/or "consisting essentially of" such features.

Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

The terms "decrease", "reduced", "reduction", or "inhibit" are all used herein to mean a decrease by a statistically significant amount. The terms "reduce," "reduction" or "decrease" or "inhibit" typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about

45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about

70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about

95%, at least about 98%, at least about 99% , or more. As used herein, "reduction" or "inhibition" encompasses a complete inhibition or reduction as compared to a reference level. "Complete inhibition" is a 100% inhibition (i.e. abrogation) as compared to a reference level. The terms "increased", "increase", "enhance", or "activate" are all used herein to mean an increase by a statically significant amount. The terms "increased", "increase", "enhance", or "activate" can mean an increase of at least 25%, at least 50% as compared to a reference level, for example an increase of at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, at least about 95%, or at least about 98%, or at least about 99%, or at least about 100%, or at least about 250% or more compared with a reference level, or at least about a 1.5-fold, or at least about a 2-fold, or at least about a 2.5-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5- fold or at least about a 10-fold increase, or any increase between 1.5-fold and 10-fold or greater as compared to a reference level. In the context of a property of the scaffold or cells seeded thereof, an "increase" is an observable or statistically significant increase in the level of said property.

The terms "individual", "subject", and "patient", are used interchangeably herein to refer to a mammalian subject for whom diagnosis, prognosis, disease monitoring, treatment, therapy, and/or therapy optimisation is desired. The mammal can be (without limitation) a human, non-human primate, mouse, rat, dog, cat, horse, or cow. In a preferred embodiment, the individual, subject, or patient is a human. An "individual" may be an adult, juvenile or infant. An "individual" may be male or female.

A "subject in need" of treatment for a particular condition can be an individual having that condition, diagnosed as having that condition, or at risk of developing that condition.

A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications or symptoms related to such a condition, and optionally, have already undergone treatment for a condition as defined herein or the one or more complications or symptoms related to said condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition as defined herein or one or more or symptoms or complications related to said condition. For example, a subject can be one who exhibits one or more risk factors for a condition, or one or more or symptoms or complications related to said condition or a subject who does not exhibit risk factors.

As used herein, the term "healthy individual" refers to an individual or group of individuals who are in a healthy state, e.g. individuals who have not shown any symptoms of the disease and/or viral infection, have not been diagnosed with the disease and/or viral infection, and/or are not likely to develop the disease and/or viral infection. Preferably said healthy individual(s) is not on medication affecting the disease or condition to be treated, and has not been diagnosed with any other disease. The one or more healthy individuals may have a similar sex, age, and/or body mass index (BMI) as compared with the test individual. Application of standard statistical methods used in medicine permits determination of normal levels of expression in healthy individuals, and significant deviations from such normal levels.

Herein the terms "control" and "reference population" are used interchangeably.

The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia.

Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogues, regardless of its size or function. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogues of the foregoing.

As used herein, the terms "polynucleotides", "nucleic acid" and "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analogue thereof. The nucleic acid can be single-stranded, doublestranded or triple-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In double or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands, and similarly for a triple-stranded nucleic acid). In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Non-limiting examples of polynucleotides may include coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA ( cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogues. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labelling component.

Minor variations in the amino acid sequences of the invention are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence(s) maintain at least 60%, at least 70%, more preferably at least 80%, at least 85%, at least 90%, at least 95%, and most preferably at least 97% or at least 99% sequence identity to the amino acid sequence of the invention or a fragment thereof as defined anywhere herein. The term homology is used herein to mean identity. As such, the sequence of a variant or analogue sequence of an amino acid sequence of the invention may differ on the basis of substitution (typically conservative substitution) deletion or insertion. Proteins comprising such variations are referred to herein as variants.

Proteins of the invention may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or nonconserved positions. Variants of protein molecules disclosed herein may be produced and used in the present invention. Following the lead of computational chemistry in applying multivariate data analysis techniques to the structure/property-activity relationships [see for example, Wold, et al. Multivariate data analysis in chemistry. Chemometrics-Mathematics and Statistics in Chemistry (Ed.: B. Kowalski); D. Reidel Publishing Company, Dordrecht, Holland, 1984 (ISBN 90-277-1846-6] quantitative activity-property relationships of proteins can be derived using well-known mathematical techniques, such as statistical regression, pattern recognition and classification [see for example Norman et al. Applied Regression Analysis. Wiley-lnterscience; 3rd edition (April 1998) ISBN: 0471170828; Kandel, Abraham et al. Computer-Assisted Reasoning in Cluster Analysis. Prentice Hall PTR, (May 11, 1995), ISBN: 0133418847; Krzanowski, Wojtek. Principles of Multivariate Analysis: A User's Perspective (Oxford Statistical Science Series, No 22 (Paper)). Oxford University Press; (December 2000), ISBN: 0198507089; Witten, Ian H. et al Data Mining: Practical Machine Learning Tools and Techniques with Java Implementations. Morgan Kaufmann; (October 11, 1999), ISBN:1558605525; Denison David G. T. (Editor) et al Bayesian Methods for Nonlinear Classification and Regression (Wiley Series in Probability and Statistics). John Wiley & Sons; (July 2002), ISBN: 0471490369; Ghose, Arup K. et al. Combinatorial Library Design and Evaluation Principles, Software, Tools, and Applications in Drug Discovery. ISBN: 0-8247-0487-8], The properties of proteins can be derived from empirical and theoretical models (for example, analysis of likely contact residues or calculated physicochemical property) of proteins sequence, functional and three-dimensional structures and these properties can be considered individually and in combination.

Amino acids are referred to herein using the name of the amino acid, the three-letter abbreviation or the single letter abbreviation. The term "protein", as used herein, includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three- letter codes for amino acid residues may be used. The 3-letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

Amino acid residues at non-conserved positions may be substituted with conservative or nonconservative residues. In particular, conservative amino acid replacements are contemplated.

A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered to be conservative. The inclusion of conservatively modified variants in a protein of the invention does not exclude other forms of variant, for example polymorphic variants, interspecies homologs, and alleles.

"Non-conservative amino acid substitutions" include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, He, Phe or Vai), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Vai, His, He or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

"Insertions" or "deletions" are typically in the range of about 1, 2, or 3 amino acids. The variation allowed may be experimentally determined by systematically introducing insertions or deletions of amino acids in a protein using recombinant DNA techniques and assaying the resulting recombinant variants for activity. This does not require more than routine experiments for a skilled person.

A "fragment" of a polypeptide comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or more of the original polypeptide.

The polynucleotides of the present invention may be prepared by any means known in the art. For example, large amounts of the polynucleotides may be produced by replication in a suitable host cell. The natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, mammalian, plant or other eukaryotic cell lines.

The polynucleotides of the present invention may also be produced by chemical synthesis, e.g. by the phosphoramidite method or the tri-ester method, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

When applied to a nucleic acid sequence, the term "isolated" in the context of the present invention denotes that the polynucleotide sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators), and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment.

In view of the degeneracy of the genetic code, considerable sequence variation is possible among the polynucleotides of the present invention. Degenerate codons encompassing all possible codons for a given amino acid are set forth below:

Amino Acid Codons Degenerate Codon

Cys TGC TGT TGY

Ser AGC AGT TCA TCC TCG TCT WSN

Thr ACA ACC ACG ACT ACN

Pro CCA CCC CCG CCT CCN

Ala GCA GCC GCG GCT GCN Gly GGA GGC GGG GGT GGN

Asn AAC AAT AAY

Asp GAC GAT GAY

Glu GAA GAG GAR

Gin CAA CAG CAR

His C AC CAT CAY

Arg AGA AGG CGA CGC CGG CGT MGN

Lys AAA AAG AAR

Met ATG ATG

He ATA ATC ATT ATH

Leu CTA CTC CTG CTT TTA TTG YTN

Vai GTA GTC GTG GTT GTN

Phe TTC TTT TTY

Tyr TAC TAT TAY

Trp TGG TGG

Ter TAA TAG TGA TRR

Asn/ Asp RAY

Glu/ GIn SAR

Any NNN

One of ordinary skill in the art will appreciate that flexibility exists when determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of the present invention.

A "variant" nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or a fragment thereof). A nucleic acid sequence or fragment thereof is "substantially homologous" (or "substantially identical") to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70%, 75%, 80%, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more% of the nucleotide bases. Methods for homology determination of nucleic acid sequences are known in the art.

Alternatively, a "variant" nucleic acid sequence is substantially homologous with (or substantially identical to) a reference sequence (or a fragment thereof) if the "variant" and the reference sequence they are capable of hybridizing under stringent (e.g. highly stringent) hybridization conditions.

As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of at least 10, preferably at least 15, and more preferably at least 20 nucleotides, preferably no more than 100 nucleotides, more preferably no more than 40 nucleotides that is hybridisable to a genomic DNA molecule, or other nucleic acid of interest.

Nucleic acid sequence hybridisation will be affected by such conditions as salt concentration (e.g. NaCI), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions are preferably employed, and generally include temperatures in excess of 30°C, typically in excess of 37°C and preferably in excess of 45°C. For universal PCR cycling, stringent temperature conditions for primer hybridisation and elongation are typically in excess of 55°C, particularly at about 60°C. For step-up PCR cycling as described herein, stringent temperature conditions for primer hybridisation and elongation typically comprise reducing the temperature to below 60°C, for example to about 50°C to about 58°C, particularly between about 52°C to about 56°C before increasing to about 60°C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. The pH is typically between 7.0 and 8.3. The combination of parameters is much more important than any single parameter.

Methods of determining nucleic acid percentage sequence identity are known in the art. By way of example, when assessing nucleic acid sequence identity, a sequence having a defined number of contiguous nucleotides may be aligned with a nucleic acid sequence (having the same number of contiguous nucleotides) from the corresponding portion of a nucleic acid sequence of the present invention. Tools known in the art for determining nucleic acid percentage sequence identity include Nucleotide BLAST (as described below).

A "fragment" of a polynucleotide of interest comprises a series of consecutive nucleotides from the sequence of said full-length polynucleotide. By way of example, a "fragment" of a polynucleotide of interest may comprise (or consist of) at least 30 consecutive nucleotides from the sequence of said polynucleotide (e.g. at least 35, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 850, 900, 950 or 1000 consecutive nucleic acid residues of said polynucleotide). A fragment may include at least one antigenic determinant and/or may encode at least one antigenic epitope of the corresponding polypeptide of interest. Typically, a fragment as defined herein retains the same function as the full-length polynucleotide. As used herein, the term "primer" refers to an isolated oligonucleotide that is capable of hybridising (also termed "annealing") with a nucleic acid and serving as an initiation site for nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer may depend on the intended use of the primer, but primers are typically at least 5 nucleotides long and more typically range from 7 to 35 nucleotides, or even more typically from 10 to 30 nucleotides, in length. In some embodiments, primers may longer, e.g., 30 to 80 nucleotides long. Primers are used to amplify a target sequence, typically by extension of the primer oligonucleotide after hybridization to the target sequence.

As used herein, the term "primer length" refers to the portion of an oligonucleotide or nucleic acid that hybridizes to a complementary "target" sequence and primes nucleotide synthesis. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridise with a template.

As used herein, the term "primer site" or "primer binding site" refers to the segment of the target nucleic acid to which a primer hybridises. A construct with presenting a primer binding site is often referred to as a "priming ready construct" or "amplification ready construct".

A probe is an isolated oligonucleotide that is used to capture or detect a target sequence to which it hybridises.

"Amplification" of a nucleic acid as used herein denotes the increase in the concentration of a particular nucleic acid sequence within a mixture of nucleic acid sequences. The use of polymerase chain reaction (PCR) is more particularly contemplated in the context of the invention.

A "nested" PCR means that two pairs of PCR primers are used for a single locus. The first pair amplifies the locus as seen in any PCR experiment. The second pair of primers ("nested primers") bind within the first PCR product and produce a second PCR product that is typically shorter than the first one. Where a tag is used in a semi-nested PCR protocol as described herein, the second PCR product may be the same length or slightly longer than the first PCR product in view of the presence of the tag sequence.

A "semi-nested" PCR is a way to get amplification of a target sequence by using two consecutive PCR runs. A first set of primers is used for the first PCR run. For the second PCR run, one of the primers used in the first run is used again and the other primer is within the target sequence. Thus, a "semi-nested" PCR using three PCR primers for a single locus. As used herein, "detecting" or "detection" refers to determining the presence or absence of a virus. This may be stated as a lower limit of detection (LOD or LLOD), which is the lowest quantity of a virus that can be distinguished from the absence of that virus (a negative control) with a stated confidence level, such as a confidence level of 90%, 95% or 99%, preferably a confidence level of 95%.

As used herein, "quantifying" or "quantification" refer to counting or measuring the amount of virus present. The amount of virus may be quantified use any appropriate units, non-limiting examples of which are well-known in the art, such as copies/mL, pfu/mL and 50% tissue culture infective dose (TCIDso). Given the methods of the invention typically relate to the use of qPCR, copies/mL, copies/cell, copies/million cells and input copies of RNA or DNA may be the preferred units for quantification.

A method of detection and/or quantification of a virus may be characterised by its "sensitivity". "Sensitivity" relates to the percentage of samples containing a particular virus that were correctly identified. "Sensitivity" is defined in the art as the number of true positives divided by the sum of true positives and false negatives.

The "specificity" of a method is defined as the percentage of samples that were correctively identified as not having a particular virus compared with an uninfected/negative control(s). That is, "specificity" relates to the number of true negatives divided by the sum of true negatives and false positives.

Typically, the sensitivity and/or specificity of a method of the invention is at least about 75%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% or at least about 97%, at least about 98%, at least about 99% or more, up to about 100%.

"Accuracy" is defined as the total number of accurately classified individuals divided by the total number of individuals subjected to characterisation. Typically, the accuracy of a method of the invention is at least about 75%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% or at least about 97%, at least about 98%, at least about 99% or more, up to about 100%.

As used herein, positive and negative predictive values (PPV and NPV respectively) are the proportions of positive and negative results that are true positive and true negative results, respectively. The PPV and NPV describe the performance of a diagnostic test or other statistical measure.

Typically, PPV and/or NPV of a method of the invention is at least about 75%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% or at least about 97%, at least about 98%, at least about 99% or more, up to about 100%.

As used herein, a compendium database (also referred to as a diversity panel) is a database comprising sequence information for a plurality of strains or subtypes of a given virus. Typically said databases are carefully curated selected from a large number of sequences that represent the diversity of the virus in question. As described herein, design of primers and/or probes using compendium databases provide advantages over the use of standard sequence alignments as per the art. Compendium databases may be supplemented with additional sequence information from recent field-based isolates of the virus in question. Examples of compendium databases include the LANL HIV-1 compendium database

(https://www.hiv.lanl. ov/content/sequence/HIV/COMPENDIUM/compendium.html) and the EQ.APOL virus diversity panel.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

Disclosure related to the various methods of the invention are intended to be applied equally to other methods, therapeutic uses or methods, the data storage medium or device, the computer program product, and vice versa.

Viruses

The invention relates to the detection and/or quantification of viruses. In particular, the invention is useful in the detection and/or quantification of viruses which have historically proven difficult to identify and/or quantify as a result of significant sequence variation between strains.

As described herein, the present invention provides a method for identifying regions within the genome of a given virus that are conserved across different strains, and using those regions to generate suitable primers and/or probes for the detection and/or quantification of said virus.

Without being bound by theory, repeat regions within viral genomes are particularly suited for targeting in methods of the invention, as those tend to be well-conserved between subtypes of a given virus. As such, identification of these regions and particularly conserved regions within repeated sequences can be used to design suitable primers and/or probes for good cross-strain/subtype detection and/or quantification.

As discussed in more detail below, the invention relates particularly to the detection and/or quantification of retroviruses/lentiviruses. Other viruses that can be detected and/or quantified according to the invention include the Hepadnaviridae family, such as Hepatitis B virus (HBV), the Picornaviridae family, such as Hepatitis A virus (HAV) and the Herpesviridae family, such as Epstein- Bar Virus (EBV).

Retroviruses and Lentiviruses

The invention relates to the detection and/or quantification of viruses, particularly to the detection and/or quantification of retroviruses/lentiviruses.

The term "retrovirus" refers to any member of the Retroviridae family of RNA viruses that encode the enzyme reverse transcriptase. The term "lentivirus" refers to a family of retroviruses. Many species are infected by lentiviruses, which are characteristically responsible for long-duration illnesses with a long incubation period. Lentiviruses are transmitted as single-stranded, positive-sense, enveloped RNA viruses. Upon entry into the target cell, the viral RNA genome is converted (reverse transcribed) into double-stranded DNA by a virally encoded reverse transcriptase that is transported along with the viral genome in the virus particle. The resulting viral DNA is then imported into the cell nucleus and integrated into the cellular DNA by a virally encoded integrase and host co-factors. Once integrated, the virus may become latent, allowing the virus and its host cell to avoid detection by the immune system. Alternatively, the virus may be transcribed, producing new RNA genomes and viral proteins that are packaged and released from the cell as new virus particles that begin the replication cycle anew.

Examples of retroviruses that may be detected and/or quantified according to the present invention include gammaretroviruses such as murine leukaemia virus (MLV) and feline leukaemia virus (FLV). Examples of lentiviruses that may be detected and/or quantified according to the present invention include Human immunodeficiency virus (HIV), Feline immunodeficiency virus (FIV), Equine infectious anaemia virus (EIAV), Simian immunodeficiency virus (SIV) and Visna/maedi virus. Preferably the invention relates to lentiviral vectors and the detection and/or quantification thereof. A particularly preferred lentivirus for detection and/or quantification according to the invention is HIV.

Two types of HIV have been characterized: HIV-1 and HIV-2. HIV-1 is the virus that was initially discovered and termed both LAV and HTLV-111. It is more virulent, more infective, and is the cause of the majority of HIV infections globally. The lower infectivity of HIV-2 compared to HIV-1 implies that fewer of those exposed to HIV-2 will be infected per exposure. Because of its relatively poor capacity for transmission, HIV-2 is largely confined to West Africa. Accordingly, the invention particularly relates to the detection and/or quantification of HIV-1.

HIV-1 can be classified into 4 groups, M, N, O and P, of which groups M, N and O, and in particular group M, are responsible for the majority of global HIV infections. Group M can be further classified into subtypes based on sequence divergence: subtypes A, B, C, D, F, G, H, J and K and hybrid circulating recombinant forms (CRFs) and unique recombinant forms (URFs). The invention may be used to detect and/or quantify any or all subtypes of HIV-1, or any further strain or subtype within these subtypes. By way of non-limiting example, the invention may relate to the detection and/or quantification of any one, two, three, four, five, six, seven, or eight of HIV-1 subtypes A, B, C, D, F, G, H, J and K in any combination, or all of said subtypes. Alternatively or in addition the invention may relate to the detection and/or quantification of one or more CRF and/or URF. A reference HIV-1 sequence is that of the HXB2 strain. The HXB2 genome sequence is given herein as SEQ ID NO: 24. Any and all references to HIV-1 herein refer equally and without reservation to the HXB2 strain and SEQ. ID NO: 24.

Retroviruses and lentiviruses have long-terminal repeats (LTRs) within their genomes, which are particularly suited to the present invention. Without being bound by theory, it is believed that these LTRs tend to be well-conserved between subtypes of a given virus, because they are untranslated and so typically not subject to immune pressure. As such, identification of these regions and particularly conserved regions within the LTRs can be used to design suitable primers and/or probes for good cross-strain/subtype detection and/or quantification. A reference HIV-1 LTR sequence is that of the HXB2 strain. The HXB2 LTR sequence is given herein as SEQ ID NO: 21. Any and all references to HIV-1 LTR herein refer equally and without reservation to the HXB2 strain LTR and SEQ ID NO: 21.

Methods and Assays for Detecting and/or Quantifying Viruses

The invention provides methods for the detection and/or quantification of viruses. Any viruses may be detected and/or quantified using the methods of the invention. Typically the methods of the invention are used to detect and/or quantify a virus as described herein, particularly HIV-1. The method of the invention is typically in vitro or ex vivo. The invention also provides assays for the detection and/or quantification of viruses. Any and all disclosure herein in relation to methods of the invention applies equally and without reservation to assays of the invention.

The present inventors are the first to appreciate that designing primers and probes merely on the basis of general sequence alignments (which is conventional in the art) results in significant discrepancies, which limit, often severely, the ability of such primers and/or probes to achieve good cross-subtype specificity. Rather, the present inventors are the first to devise a paradigm shift for viral detection and/or quantification, and to demonstrate that this entirely new approach can be used to generate primers and/or probes that can achieve improved cross-subtype specificity compared with more conventionally designed primers and/or probes. In particular, for any given virus, the present invention relates to the use of carefully curated sequence information to identify regions which are conserved across different strains or subtypes, and generating primers and/or probe sequences based on those conserved regions to detect and/or quantify the virus. In the case of HIV-1, the NCBI/LANL

HIV-1 compendium sequence database (accessible here:

.him I) may preferably be used.

Accordingly, the invention provides a new approach for designing primer and/or probe sequences useful in the detection and/or quantification of viruses, particularly the invention provides a new approach for designing primer and/or probe sequences useful in the detection and/or quantification of viruses with multiple strains or subtypes at high levels of cross-strain specificity. In particular, the invention provides a method of designing primer and/or probe sequences, which method comprises the steps of: (a) identifying an nucleotide region that is conserved across multiple different strains or subtypes of a virus (e.g. at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes) by aligning a panel of a plurality of different strains or subtypes of said virus (e.g. a panel of at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes); and (b) designing one or more primer and/or probe sequences that are suitable for the preferred amplification method based on the identified nucleotide region, wherein said primer and/or probe sequences are specific to the identified nucleotide region.

The panel of a plurality of different viral strains or subtypes in step (a) may comprise sequence information for recent circulating strains or subtypes of the virus in question.

The panel of a plurality of different viral strains or subtypes in step (a) may be interrogated using commercially available software, such as allelelD, primer 3 and the IDT oligoanalyzer to identify nucleotide regions that are conserved across multiple different strains or subtypes, particularly regions of maximal homology across the diverse strains that in combination provide good primers and/or probes for highly specific and sensitive detection and/or amplification. Step (b) may optionally comprise conducting a BLAST analysis following design of the one or more primer and/or probe sequence to verify that the designed sequences are specific for the virus in question.

In addition, the invention provides a new approach for the detection and/or quantification of viruses, particularly the invention provides a new approach for the detection and/or quantification of viruses with multiple strains or subtypes at high levels of cross-strain specificity. In particular, the invention provides a method of detecting and/or quantifying a virus, which method comprises the steps of: (a) identifying an nucleotide region that is conserved across multiple different strains or subtypes of a virus (e.g. at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes) by aligning a panel of a plurality of different strains or subtypes of said virus (e.g. a panel of at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes); (b) designing one or more primer and/or probe sequences that are suitable for the preferred amplification method based on the identified nucleotide region, wherein said primer and/or probe sequences are specific to the identified nucleotide region; and (c) detecting and/or quantifying the virus within a biological sample by amplifying viral nucleic acid using the one or more primer and/or probe sequence.

The methods of the invention may use polymerase chain reaction (PCR) to amplify the viral nucleic acid. Any suitable form of PCR may be used, including standard PCR, droplet digital PCR (ddPCR), reverse transcription PCR (RT PCR), quantitative PCR (qPCR, also known as real-time PCR) and reverse transcription quantitative PCR (RT-qPCR). Alternatively, isothermal amplification approaches such as Loop-Mediated-lsothermal Amplification (LAMP), CRISPR-based isothermal amplification, nucleic acid sequence-based amplification (NASBA), strand-displacement amplification (SDA) and/or recombinase polymerase amplification (RPA) strategies may be used according to the present invention. Such techniques typically insert engineered primer-sites into target identified according to the new approach of the invention as described herein, and use primers against the resulting engineered targets for amplification. These are standard techniques that are well-known in the art and within the routine skill of one of ordinary skill in the art.

The invention therefore provides a method of detecting and/or quantifying a virus, which method comprises the steps of: (a) identifying an nucleotide region that is conserved across multiple different strains or subtypes of a virus (e.g. at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes) by aligning a panel of a plurality of different strains or subtypes of said virus (e.g. a panel of at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes); (b) designing one or more primer and/or probe sequences that are suitable for the preferred amplification method based on the identified nucleotide region, wherein said primer and/or probe sequences are specific to the identified nucleotide region; and (c) detecting and/or quantifying the virus within a biological sample by amplifying viral nucleic acid by PCR (e.g. qPCR), LAMP, CRISPR-based or other isothermal amplification using the one or more primer and/or probe sequence.

Any suitable primer and/or probe sequences may be used in methods of the present invention. Suitable design constraints and exemplary sequences are described herein. In the context of retroviruses/lentiviruses (e.g. HIV-1), the invention provides a method of detecting and/or quantifying a retrovirus/lentivirus (e.g. HIV-1), which method comprises the steps of: (a) identifying an LTR region that is conserved across multiple different strains or subtypes of a virus (e.g. at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes) by aligning a panel of a plurality of different strains or subtypes of said virus (e.g. a panel of at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes); (b) designing one or more primer and/or probe sequences that are suitable for the preferred amplification method based on the identified LTR region, wherein said primer and/or probe sequences are specific to the identified LTR region; and (c) detecting and/or quantifying the virus within a biological sample by amplifying viral nucleic acid by PCR (e.g. qPCR), LAMP, CRISPR-based or other isothermal amplification using the one or more primer and/or probe sequence. The primer and/or probe sequence used in said method may be designed as described herein.

In some preferred embodiments, the methods of the invention include a qPCR reaction (qPCR or RT-qPCR). The use of qPCR or RT-qPCR is advantageous as the amplified nucleic acids are detected as the reaction progresses in "real time".

In particular, as exemplified herein using HIV-1, the invention provides a method of detecting and/or quantifying a virus, which method comprises the steps of: (a) amplifying viral nucleic acid in a biological sample with PCR using a first and second primer for a highly conserved region of the pathogen such as long terminal repeat (LTR) region of lentiviruses, wherein said first primer is linked to a tag sequence; (b) subjecting the amplified nucleic acid from step (a) to another amplification with PCR using the second primer and a primer for the tag sequence; and (c) detecting and/or quantifying the nucleic acid that was amplified through steps (a) and (b), wherein the detected/quantified nucleic acid correlates with the number of copies of the virus genome. The primer and/or probe sequence used in said method may be designed as described herein.

The first and second primers in step (a) of said method typically hybridise to different regions within the LTR sequence. This allows the viral nucleic acid to be amplified using semi-nested PCR. This semi-nested format of the method allows for the use of only three regions and increases cross-subtype coverage. This semi-nested format typically also allows for target enrichment, making it particularly useful with limited samples (e.g. low volume and/or low concentration samples). Typically a seminested format also increases assay sensitivity compared with non-nested or nested assay types.

The methods of the invention may be used to detect and/or quantify total viral RNA and/or DNA. Thus, the methods of the invention may be used to detect and/or quantify viral RNA and/or DNA that is (i) extracellular; (ii) intracellular but not integrated into the host cell DNA (otherwise referred to as unintegrated); and/or (iii) intracellular and integrated into the host cell DNA, typically all of (i), (ii) and (iii). By allowing the detection and/or quantification of total intracellular viral RNA and/or DNA, the methods of the invention have the potential to detect virus earlier than conventional assays which rely on the detection of extracellular viral nucleic acid.

The invention also provides assays and methods for the detection of integrated virus specifically. Any integrated viruses may be detected and/or quantified using the methods of the invention. Typically the methods of the invention are used to detect and/or quantify an integrated virus as described herein, particularly HIV-1. The method of the invention is typically in vitro or ex vivo. The invention also provides assays for the detection and/or quantification of integrated viruses. Any and all disclosure herein in relation to methods of the invention for the detection and/or quantification of integrated viruses applies equally and without reservation to assays of the invention for the detection and/or quantification of integrated viruses.

The invention therefore provides a method of detecting and/or quantifying an integrated virus, which method comprises the steps of: (a) identifying an nucleotide region that is conserved across multiple different strains or subtypes of a virus (e.g. at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes) by aligning a panel of a plurality of different strains or subtypes of said virus (e.g. a panel of at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes); (b) designing one or more primer and/or probe sequences that are suitable for the preferred amplification method based on the identified nucleotide region, wherein said primer and/or probe sequences are specific to the identified nucleotide region; and (c) detecting and/or quantifying the virus within a biological sample by amplifying viral nucleic acid by PCR (e.g. qPCR), LAMP, CRISPR-based or other isothermal amplification using the one or more primer and/or probe sequence and a primer for an Alu sequence. A first primer for the viral nucleotide region may be linked to a tag sequence to facilitate detection and/or quantification of the integrated virus. Any suitable primer and/or probe sequences may be used in methods of the present invention. Suitable design constraints and exemplary sequences are described herein.

In the context of retroviruses/lentiviruses (e.g. HIV-1), the invention provides a method of detecting and/or quantifying a retrovirus/lentivirus (e.g. HIV-1), which method comprises the steps of: (a) identifying an LTR region that is conserved across multiple different strains or subtypes of a virus (e.g. at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes) by aligning a panel of a plurality of different strains or subtypes of said virus (e.g. a panel of at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes); (b) designing one or more primer and/or probe sequences that are suitable for the preferred amplification method based on the identified LTR region, wherein said primer and/or probe sequences are specific to the identified LTR region; and (c) detecting and/or quantifying the virus within a biological sample by amplifying viral nucleic acid by PCR (e.g. qPCR), LAMP or CRISPR-based isothermal amplification using the one or more primer and/or probe sequence and a primer for an Alu sequence. A first primer for the viral LTR sequence may be linked to a tag sequence to facilitate detection and/or quantification of the integrated virus. The primer and/or probe sequence used in said method may be designed as described herein. The primer for the viral LTR (e.g. HIV-1 LTR) may preferably hybridise to the U5 or R region of the 5' LTR. The U5 region of the LTR (e.g. HIV-1 LTR) is typically defined as comprising or consisting of approximately nucleotide positions 570-633 of the viral (e.g. HIV-1 genome). The R region of the LTR (e.g. HIV-1 LTR) is typically defined as comprising or consisting of approximately nucleotide positions 470-570 of the viral (e.g. HIV-1 genome).

Again, these methods of the invention typically use polymerase chain reaction (PCR) to amplify the viral nucleic acid, as described herein. In some preferred embodiments, the methods of the invention include a qPCR reaction (qPCR or RT-qPCR). Alternatively, methods of the invention may amplify the viral nucleic acid using techniques such as LAMP, CRISPR-based isothermal amplification, NASBA, SDA and/or RPA as described herein.

The invention therefore provides a method of detecting and/or quantifying an integrated virus, which method comprises the steps of: (a) amplifying viral nucleic acid in a biological sample with PCR using a primer for an Alu sequence and a first primer for a viral LTR sequence, wherein said first primer for a viral LTR sequence is linked to a tag sequence; (b) subjecting the amplified nucleic acid from step (a) to another amplification with PCR using a second primer for the viral LTR sequence and a primer for the tag sequence; and (c) detecting and/or quantifying the nucleic acid that was amplified through steps (a) and (b), wherein the detected/quantified nucleic acid correlates with the number of copies of the integrated virus genome. The primer and/or probe sequence used in said method may be designed as described herein.

The use of the same primer for a viral conserved sequence (e.g. an LTR) in steps (a) and (b) of said method of detecting and/or quantifying an integrated virus allows the viral nucleic acid to be amplified using semi-nested PCR. The advantages of such a semi-nested format are described herein.

The primer and/or probe sequences of the invention may be used in a method of detecting and/or quantifying a virus, particularly HIV-1. Said method may comprise or consist of carrying out PCR (particularly qPCR) using one or more oligonucleotides primers of the invention and/or one or more oligonucleotide probe of the invention. Preferably said method comprises or consists of carrying out PCR using both oligonucleotide primers and probes of the invention, such as a set of oligonucleotides as defined herein. Exemplary target regions, primer and probe sequences for other amplification techniques may be readily determined by one of ordinary skill in the art using the disclosure herein. Non-limiting examples of target regions, primer and probe sequences within HIV-1 LTR for LAMP amplification are also described. Methods of the invention may be used to detect and/or quantify total viral RNA and/or DNA, as described herein. Such methods of the invention are typically in vitro or ex vivo. The invention also provides assays for the detection and/or quantification of viruses using the primers and/or probes of the invention. Any and all disclosure herein in relation to methods of the invention for the detection and/or quantification of viruses using the primers and/or probes of the invention applies equally and without reservation to assays of the invention for the detection and/or quantification of viruses using the primers and/or probes of the invention.

Again, these methods of the invention may use polymerase chain reaction (PCR) to amplify the viral nucleic acid, as described herein. In some preferred embodiments, the methods of the invention include a qPCR reaction (qPCR or RT-qPCR). Said methods may comprise one round of amplification (e.g. as exemplified in Figure 2b), or two-rounds (e.g. as exemplified in Figure 2c). Said methods may comprise qPCR or RTqPCR; and/or nested or semi-nested PCR, as described herein. Alternatively, other amplification techniques, such as LAMP, CRISPR-based isothermal techniques, NASBA, SDA and/or RPA may be used.

In some preferred embodiments, the sensitivity, specificity, positive predictive value, negative predictive value and/or accuracy of a method of the invention may each independently be at least about 85%, at least about 90%, at least about 93%, at least about 95% or more, up to about 100%. By way of non-limiting example, the sensitivity of a method of the invention may be at least about 95% , the specificity of a method of the invention may be at least about 90%, the positive predictive value of a method of the invention may be at least about 85%, the negative predictive value of a method of the invention may be at least about 95%, and the accuracy of a method of the invention may be at least about 90%.

In some embodiments, the sensitivity, specificity, positive predictive value, negative predictive value and/or accuracy of a method of the invention may each independently be increased by at least about 10 fold to at least about 100 fold. Preferably, the sensitivity, specificity, positive predictive value, negative predictive value and/or accuracy of a method of the invention using cell lysates as a sample and a step-up protocol as described herein may each independently be increased by at least about 10 fold to at least about 100 fold compared with conventional method.

A method of the invention may be used to detect and/or quantify low concentrations of virus. Thus, a method of the invention may have a lower limit of detection (LLOD) of from about 10 copies/mL to about 100 copies/mL, such as from about 15 copies/mL to about 90 copies/mL, from about 20 copies/mL to about 90 copies/mL, from about 25 copies/mL to about 90 copies/mL, from about 50 copies/mL to about 90 copies/mL, from about 60 copies/mL to about 90 copies/mL, from about 70 copies/mL to about 90 copies/mL, or from about 80 copies/mL to about 90 copies/mL. A method of the invention may have a lower limit of detection (LLOD) of about 80 copies/mL, 81 copies/mL, 82 copies/mL, 83 copies/mL, 84 copies/mL, 85 copies/mL, 86 copies/mL, 87 copies/mL, 88 copies/mL, 89 copies/mL, 90 copies/mL. A method of the invention may have a lower limit of detection (LLOD) of about 15 copies/mL, 16 copies/mL, 17 copies/mL, 18 copies/mL, 19 copies/mL, or 20 copies/mL. The LLOD may be determined for a given confidence level, for example at 50% confidence, 60% confidence, 70% confidence, 80% confidence, 85% confidence, 90% confidence, 91% confidence, 92% confidence, 93% confidence, 94% confidence, 95% confidence, 96% confidence, 97% confidence, 98% confidence or 99% confidence. The LLOD may vary depending on the confidence level. By way of non-limiting example, the LLOD may be from about 60 copies/mL to about 90 copies/mL, such as from about 80 copies/mL to about 90 copies/mL, particularly about 88 copies/mL at 95% confidence. By way of a further non-limiting example, the LLOD may be from about 15 copies/mL to about 50 copies/mL, such as from about 15 copies/mL to about 25 copies/mL, particularly about 17 copies/mL at 95% confidence.

The methods of the invention may be used to detect and/or quantify low concentration of viral nucleic acid. Preferably, a method of the invention may be used to detect and/or quantify ultra-low concentrations of virus. As used herein, the term "ultra-low concentration detection" refers to detection of DNA or RNA at a concentration of 1 copy /mL or less. Such methods may be described as "ultra-sensitive".

The methods of the invention involves detecting and/or quantifying the amplified viral nucleic acid. For two-round methods, these involve detecting and/or quantifying the viral nucleic that was amplified through steps (a) and (b). The detected and/or quantified nucleic acid may correlate with the number of copies of the viral genome, particularly for qPCR-based methods.

Semi-nested and nested version of the methods of the invention (e.g. the two-round methods described herein) are particularly useful when non-nested forms of the method do not achieve the desired sensitivity (e.g. ultra-sensitivity) and/or when target enrichment is required. Target enrichment, and hence a semi-nested or nested method (e.g. a two-round method as described herein) may be required for viral load testing.

Non-nested methods of the invention (e.g. the one round methods described herein) are typically used when detecting and/or quantifying viral nucleic acid (e.g. DNA or RNA) from within crude-lysates of patient cells (e.g. peripheral blood mononuclear cells or other cellular locations where the virion is sequestered) and concentrated and is detectable even when viral RNA is undetectable in extracellular materials. By way of non-limiting example, in the case of HIV-1, a non-nested method may be used to detect and/or quantify HIV-1 DNA within PBMC lysates when HIV-1 RNA is undetectable in the plasma of a patient, e.g., during or as a result of cART. Thus, the one round methods of the invention may be clinically useful as an quick, cheap and easy means of detecting, quantifying and/or estimating the latent viral reservoir, and so represent a clinically advantageous alternative to existing viral (e.g. HIV-1) load testing, which rely on plasma RNA-based assays.

Cell-associated viral DNA (e.g. HIV-1 DNA) quantification has been shown to be a better prognostic indicator of disease status compared with viral load monitoring. Accordingly, quantification of viral DNA (e.g. HIV-1 DNA) by a method of the invention correlate with CD4/CD8 T cell ratio in HIV-infected PBMCs.

The advantages and potential applications of different PCR-based methods of the invention are set out in Figure 1.

The methods of the invention may use the same cycling parameters for steps (a) and (b) (also referred to as universal cycling). Alternatively, the methods of the invention may use different cycling parameters for steps (a) and (b).

Step (a) may comprise or consist of from about 10 to about 15 cycles. Step (b) may comprise or consist of from about 30 to about 50 cycles. Step (a) may comprise or consist of from about 10 to about 15 cycles and step (b) may comprise or consist of from about 30 to about 50 cycles. Step (a) and/or step (b) may also comprise a polymerase activation step. Wherein said method involves RT PCR or RT-qPCR, step (a) may also comprise a reverse transcription step.

A method of the invention may comprise diluting the amplified nucleic acid from step (a) prior to carrying out the amplification of step (b). Typically such dilutions are from about a two-fold dilution to about a 100-fold dilution, such as from about a two-fold dilution to about a 20-fold dilution, or from about a five-fold dilution to about a 15-fold dilution. Preferably the amplified nucleic acid from step (a) is diluted about 10-fold prior to carrying out the amplification of step (b).

Methods with one-round amplification may comprise or consist of from about 30 to about 50 amplification cycles. Said methods may optionally comprise from about 0 to about 15 preamplification cycles. Alternatively or in addition, said methods may also comprise a polymerase activation step. Wherein said method involves RT PCR or RT-qPCR, said methods may also comprise a reverse transcription step.

The variability of the HIV-1 genome makes it very challenging to find three well-conserved regions of 18-35 nucleotides (two for the primers and one for the internal probe) within 200 base pairs of each other, for use with TaqMan PCR. Step-up amplification allow for less stringent annealing at lower initial temperatures, followed gradually by higher more stringent annealing temperatures. Step down amplification allow for more stringent annealing at higher initial temperatures, followed gradually by lower, less stringent annealing temperatures. Accordingly, the methods of the invention may use step-up PCR (also referred to as touch-up PCR) or step-down PCR (also referred to as touchdown PCR). In step-down PCR the annealing temperature of the initial cycle(s) is usually a few degrees (3-5 °C) above the T_m of the primers used, while at the later cycles, it is a few degrees (3-5 °C) below the primer T_m. Preferably step-up PCR is used. In step-up PCR, the annealing temperature of the initial cycle(s) is usually a few degrees (2-8 °C) below the T_m of the primers used, while at the later cycles, it is a few degrees (2-8 °C) above the primer T_m. As exemplified herein, step-up PCR can enable binding of primers across the LTR regions with improved cross-subtype specificity and sensitivity compared with universal cycling. Step-up ( or touch-up) PCR may be used with any methods of the invention, and is used in combination with a semi-nested format if target enrichment is described herein. Methods of the invention may use hybrid step-up PCR. The term "hybrid step-up PCR" encompasses PCR in which the annealing temperature is increased in equal incremental steps throughout the amplification cycles, rather than having a lower initial annealing temperature and subsequently jumping to a higher annealing temperature. Unless expressly stated otherwise, all disclosures herein to step up PCR apply equally and without reservation to hybrid step-up PCR. By way of non-limiting example, in the exemplary cycling protocols described herein, a hybrid step-up amplification protocol may comprise or consist of an initial activation step of 95 °C for 1-15 minutes, followed by 1-15 pre-amplification cycles of 92-94 °C for 1-20 seconds, incrementally increasing the annealing temperature from 52 °C up to 55-65 °C in equal steps over the course of the preamplification cycles, followed by 30-45 amplification cycles of 92-95 °C for 20 seconds, 56 °C (or 1-15 °C below the ideal annealing temperature) for 1-10 seconds, 57-66 °C for 2-60 seconds. Alternatively, a hybrid step-up amplification protocol may comprise 30-45 amplification cycles in which the annealing temperature is incrementally increased in equal steps from 56-66 °C over the course of the amplification cycles.

The invention also provides unique combinations of PCR cycling parameters that have been designed and demonstrated to facilitate detection and/or quantification of viral nucleic acids at low concentrations, and/or with high sensitivity, specificity, positive predictive value, negative predictive value and/or accuracy as described herein. Any combination of cycling parameters according to the invention may be used with any method of the invention as described herein.

For a one round amplification for DNA targets (such as that exemplified in Figure 2b), a "step- up" cycling protocol may comprise or consist of an initial activation step selected based on the activation parameters of the polymerase enzyme being used, for example 94-96°C for 1-20 minutes; 1-15 pre-amplification cycles of 92-97°C for 1-25 seconds, 50-54°C (or 1-15°C below the ideal annealing temperature) for 1-15 seconds and 50-65°C for 1-90 seconds, followed by 25-50 amplification cycles of 93-97°C for 2-25 seconds, 54-58°C (or 1-15°C below the ideal annealing temperature) for 1-15 seconds, 50-65°C for 1-90 seconds. For example, a one round amplification "step-up" cycling protocol may comprise or consist of an initial activation step of 95°C for 1-15 minutes, 1-15 pre-amplification cycles of 92-94°C for 1-20 seconds, 52°C (or l-10°C below the ideal annealing temperature) for 1-10 seconds and 55-65°C for 2-60s, followed by 30 to 45 amplification cycles of 92 to 95°C for 20 seconds, 56°C (or 1-15°C below the ideal annealing temperature) for 1-10 seconds, 55-60°C for 2-60s. A preferred one round amplification "step-up" cycling protocol may comprise or consist of an initial activation step of 95°C for 15 minutes, 3 pre-amplification cycles of 94°C for 20 seconds, 52°C (or 8°C below the ideal annealing temperature) for 10 seconds and 60°C for 1 minute, followed by 40 amplification cycles of 94°C for 20 seconds, 56°C (or 4°C below the ideal annealing temperature) for 10 seconds, 60°C for 1 minute.

For a one round amplification for RNA targets, a "step-up" cycling protocol may comprise or consist of an initial reverse transcription step of 40-60°C for 2-20 minutes, followed by a polymerase activation step selected based on the activation parameters of the polymerase enzyme being used, for example of 94-96°C for 1-20 minutes, followed by 25-50 amplification cycles of 93-97°C for 1-25 seconds, 50-55°C->57-63°C* (arrow and asterisk indicate that the annealing temperature is increased from 50-55°C->57-62°C in equal increments across the 25-50 cycles) and finally 65-76°C for 10-90 seconds. For example, for a one round amplification for RNA targets, a "step-up" cycling protocol may comprise or consist of an initial reverse transcription step of 45-55°C for 5-15 minutes, followed by a polymerase activation step of 94-96°C for 1-3 minutes, followed by 30-50 amplification cycles of 94- 96°C for 5-15 seconds, 51-53°C->59-61°C* (arrow and asterisk indicate that the annealing temperature is increased from 51-53°C->59-61°C in equal increments across the 30-50 cycles) and finally 70-74°C for 30-90 seconds. A preferred one round amplification "step-up" for RNA targets cycling protocol may comprise or consist of an initial reverse transcription step of 50°C for 10 minutes, followed by a "step up" cycling protocol with an enzyme activation step of 95°C for 2 minutes; 40 amplification cycles of 95°C for 10 seconds, 52°C -> 60°C* (the arrow and asterisk indicate that the annealing temperature is increased from 52°C to 60°C in equal increments across 40 cycles) for 10 seconds and finally 72°C for 1 minute.

For a method comprising a two-round amplification (such as the semi-nested format described herein and exemplified in Figure 2c), the first round cycling parameters may comprise or consist of a polymerase activation step selected based on the activation parameters of the polymerase enzyme being used, for example of 94-96°C for 1-20 minutes, followed by 1-15 (e.g. 1-12) amplification cycles of 90-97°C for 1-25 seconds, 51-53°C for 1-25 seconds and 59-61°C* for 5-90 seconds, followed by 5- 20 amplification cycles of 90-97°C for 1-25 seconds, 55-57°C for 1-25 seconds and 59-61°C* for 5-90 seconds. For example, for a method comprising a two-round amplification (such as the semi-nested format described herein and exemplified in Figure 2c), the first round cycling parameters may comprise or consist of a polymerase activation step of 94-96°C for 1-3 minutes, followed by 3-7 amplification cycles of 94-96°C for 15-25 seconds, 51-53°C for 5-14 seconds and 59-61°C* for 5-60 seconds, followed by 5-10 amplification cycles of 94-96°C for 15-25 seconds, 55-57°C for 5-15 seconds and 59-61°C* for 30-90 seconds. For methods using RT-qPCR, the polymerase activation step may be preceded by a reverse transcriptase step of 40-60°C for 2-20 minutes, such as an initial reverse transcription step of 45-55°C for 10-20 minutes. For a two-round amplification, a preferred first round cycling parameters may comprise or consist of a polymerase activation step of 95°C for 2 minutes; five amplification cycles of 95°C for 20 seconds; 52°C for 10 seconds and 60°C for 1 minute; followed by seven cycles of 95°C for 20 seconds, 56°C for 10 seconds, and 60°C for 1 minute. For methods using RT-qPCR, the polymerase activation step may be preceded by a reverse transcriptase step of 50°C for 5-15 minutes.

For a method comprising a two-round amplification, the second round cycling parameters may comprise or consist of an initial activation step of 94-96°C for 1-20 minutes, 1-15 pre-amplification cycles of 92-97°C for 1-25 seconds, 50-54°C (or 1-15°C below the ideal annealing temperature) for 1- 15 seconds and 50-65°C for 1-90 seconds, followed by 25-50 amplification cycles of 93-97°C for 2-25 seconds, 54-58°C (or 1-15°C below the ideal annealing temperature) for 1-15 seconds, 50-65°C for 1- 90 seconds. For example, for a method comprising a two-round amplification, the second round cycling parameters may comprise or consist of an initial activation step of 95°C for 1-15 minutes, 1-10 pre-amplification cycles of 92-94°C for 1-20 seconds, 52°C (or l-10°C below the ideal annealing temperature) for 1-10 seconds and 55-65°C for 2-60s, followed by 30 to 45 amplification cycles of 92 to 95°C for 20 seconds, 56°C (or 1-15°C below the ideal annealing temperature) for 1-10 seconds, 55- 60°C for 2-60s. For a two-round amplification, a preferred second round cycling parameters may comprise or consist of an initial activation step of 95°C for 15 minutes, 3 pre-amplification cycles of 94°C for 20 seconds, 52°C (or 8°C below the ideal annealing temperature) for 10 seconds and 60°C for 1 minute, followed by 40 amplification cycles of 94°C for 20 seconds, 56°C (or 4°C below the ideal annealing temperature) for 10 seconds, 60°C for 1 minute.

Thus, in some particularly preferred embodiments of methods comprising two-round amplification: (i) the first round cycling parameters comprise or consist of a polymerase activation step of 95°C for 2 minutes; five amplification cycles of 95°C for 20 seconds; 52°C for 10 seconds and 60°C for 1 minute; followed by seven cycles of 95°C for 20 seconds, 56°C for 10 seconds, and 60°C for 1 minute; and (ii) the preferred second round cycling parameters comprise or consist of an initial activation step of 95°C for 15 minutes, 3 pre-amplification cycles of 94°C for 20 seconds, 52°C (or 8°C below the ideal annealing temperature) for 10 seconds and 60°C for 1 minute, followed by 40 amplification cycles of 94°C for 20 seconds, 56°C (or 4°C below the ideal annealing temperature) for 10 seconds, 60°C for 1 minute. For preferred two-round methods using RT-qPCR, the polymerase activation step may be preceded by a reverse transcriptase step of 50°C for 5-15 minutes.

As described herein, as well as PCR-based methods which require cycling to different temperatures to facilitate amplification of the nucleic acid, the invention also provides isothermal methods, in which primers and/or probes to identified conserved nucleotide regions within a virus are used to amplify viral nucleic acid at a constant temperature. Such techniques include LAMP, CRISPR- based amplification, NASBA, SDA and/or RPA. Isothermal techniques may be preferred in circumstances where access to equipment such as thermocyclers (which are required for PCR) are not readily available, and/or to reduce costs. Such isothermal techniques are also suitable for point-of- care (POC) testing.

One of ordinary skill in the art will be readily able to determine suitable conditions and parameters for carrying out isothermal amplification, including selection of a suitable polymerase, temperature and incubation time. By way of non-limiting example, for LAMP: (i) a strand-displacing DNA polymerase (e.g. Bst or Bsm polymerase) must be used; (ii) a temperature of from about 60°C to about 65°C may be used; and/or (iii) a reaction time of about 1-60 minutes, preferably about 1-30 minutes, more preferably about 1-15 minutes, such as about 5-10 minutes may be used. By way of further non-limiting example, for SDA: (i) a temperature of from about 37°C to about 60°C may be used; (ii) a reaction time of about 1-60 minutes, preferably about 30-60 minutes, such as about 30- 45 minutes may be used; and/or (iii) a strand-displacing polymerase and a nicking endonuclease may be used. By way of a further non-limiting example, for RPA: (i) a temperature of from about 37°C to about 42°C may be used; (ii) a reaction time of about 1-60 minutes, preferably about 1-30 minutes, more preferably about 1-20 minutes, such as about 5-10 minutes may be used; and/or (iii) a stranddisplacing polymerase, a recombinase and a single-strand DNA binding protein may be used. By way of a further non-limiting example, for NASBA: (i) a temperature of about 60-65°C may be used for priming, followed by a temperature of about 40-45°C, such as about 42°C may be used; (ii) a reaction time of about 60-120 minutes; and/or (iii)a reverse transcriptase, RNAase H and T7 RNA polymerase may be used.

Such isothermal techniques may be used to amplify viral DNA or RNA. By way of non-limiting example, LAMP, RPA and/or SDA may be used to amplify viral RNA by adding a reverse transcriptase to the reaction (e.g. in the master mix). NASBA may be used to amplify DNA using known modifications to the technique.

Whilst isothermal techniques by definition are carried out at a constant temperature, the invention also encompasses the use of pseudo-isothermal techniques, particularly those which involve a step-up. In particular, the invention encompasses carrying out a pseudo-isothermal technique in which an initial portion of the reaction time is carried out at a lower temperature, followed by the remainder of the reaction time being carried out at a higher temperature. All disclosure herein in relation to isothermal techniques and methods comprising isothermal amplification apply equally and without reservation to such pseudo-isothermal techniques.

Any appropriate means for detecting and/or quantifying the amplified nucleic acid may be used. Suitable techniques are known in the art and are within the routine practice of one of ordinary skill in the art. In embodiments of the invention wherein qPCR or RT-qPCR are used to detect and/or quantify the amplified nucleic acids, typically detection of the amplified nucleic acids comprises the use of (i) non-specific dyes (e.g. fluorescent dyes) that intercalate with any double-stranded DNA, or (ii) sequence-specific oligonucleotide probes that are labelled with a reporter (e.g. a fluorescent reporter) which permits detection only after hybridisation of the probe with its complementary sequence to quantify the amplified nucleic acid.

Preferably, when present in method of the invention, the PCR of step (b) is performed in the presence of at least one detectable probe that specifically hybridises with the viral nucleic acid amplified, wherein the hybridisation of the probe allows for the detection and/or quantification of the virus. Any suitable probe may be used. Non-limiting examples are described herein.

In some embodiments, the invention relates to CRISPR-based diagnostics. In particular, CRISPR-based techniques may be used to detect and/or quantify the virus within a biological sample which has been amplified by LAMP or another isothermal amplification using the one or more primer and/or probe sequence. Accordingly, the invention provides a method of detecting and/or quantifying a virus, which method comprises the steps of: (a) identifying an nucleotide region that is conserved across multiple different strains or subtypes of a virus (e.g. at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes) by aligning a panel of a plurality of different strains or subtypes of said virus (e.g. a panel of at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes); (b) designing one or more primer and/or probe sequences that are suitable for the preferred amplification method based on the identified nucleotide region, wherein said primer and/or probe sequences are specific to the identified nucleotide region; and (c) amplifying viral nucleic acid by LAMP or another isothermal amplification technique using the one or more primer and/or probe sequence and detecting and/or quantifying amplified viral nucleic acid using CRISPR.

In the context of retroviruses/lentiviruses (e.g. HIV-1), the invention provides a method of detecting and/or quantifying a retrovirus/lentivirus (e.g. HIV-1), which method comprises the steps of: (a) identifying an LTR region that is conserved across multiple different strains or subtypes of a virus (e.g. at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes) by aligning a panel of a plurality of different strains or subtypes of said virus (e.g. a panel of at least 2, 3, 4, 5, 10, 15, 20 or more strains or subtypes); (b) designing one or more primer and/or probe sequences that are suitable for the preferred amplification method based on the identified LTR region, wherein said primer and/or probe sequences are specific to the identified LTR region; and (c) amplifying viral nucleic acid by LAMP or another isothermal amplification technique using the one or more primer and/or probe sequence and detecting and/or quantifying amplified viral nucleic acid using CRISPR.

In particular, as exemplified herein using HIV-1, the invention provides a method of detecting and/or quantifying a virus, which method comprises the steps of: (a) amplifying viral nucleic acid in a biological sample with LAMP or another isothermal amplification technique using two or more primers for the HIV-1 LTR; and (b) detecting and/or quantifying the nucleic acid that was amplified in step (a) using CRISPR. The primer sequences used in said method may be designed as described herein. By way of none-limiting example, amplification by LAMP may comprise the use of a forward internal primer (FIP typically comprising the Flc and F2 regions), a forward outer primer (F3), a backward internal primer (BIP typically comprising Bic and B2 regions) and backward outer primer (B3) and optionally a forward loop primer (LF) and/or backward loop primer (LB). Exemplary targets within HIV-1 LTR for LAMP primers and exemplary LAMP primers are described herein. crRNAs for use in CRISPR-based detection and/or quantification of an amplified viral nucleic acid are also typically designed to target the amplified viral nucleic acid. The specific crRNA sequence for use in CRISPR-based detection and/or quantification of an amplified viral nucleic acid may depend on the particular enzyme used for CRISPR-based detection. The GC content of a crRNA may be from about 40% to about 80% in order to stabilise the RNA-DNA duplex which forms during CRISPR. The length of the guide sequence within the crRNA is typically between 17-24 nucleotides, such as about 20 nucleotides. General design principles for crRNA for CRISPR-based diagnostics are known in the art, for example as described in Kaminski et al. (Nat. Biomed. Eng. 2021, 5:643-656, particularly Table 1), which is herein incorporated by reference in its entirety.

Multiple different CRISPR-based detection and/or quantification strategies are known in the art. Again, these are reviewed in Kaminski etal. (Nat. Biomed. Eng. 2021, 5:643-656, particularlyTable 1), which is herein incorporated by reference in its entirety. Selection of a suitable CRISPR-based strategy is within the routine practice of one of ordinary skill in the art.

The invention also provides unique combinations of reagents, e.g. a master-mix that have been designed and demonstrated to facilitate detection and/or quantification of viral nucleic acids at low concentrations, and/or with high sensitivity, specificity, positive predictive value, negative predictive value and/or accuracy as described herein. Any combinations of reagents, e.g. a master-mix, according to the invention may be used with any method of the invention as described herein. By way of non-limiting example, each reaction in a method of the invention may comprises a concentration of lOpg/mL of tRNA (particularly yeast tRNA). Optionally this may be achieved by using a master-mix which comprises a concentration of lOpg/mL of tRNA (particularly yeast tRNA), as described herein. The specific ingredients of a given master mix will typically depend on the amplification technique and protocol being used, e.g. qPCR (two-round or one-round), LAMP or CRISPR-based amplification. By way of non-limiting example, a PCR master mix will contain a suitable polymerase (e.g. TaqMan). The primers and/or probes present in a PCR master mix will depend on whether a one-round or two-round method is required, as described herein. By way of a further non-limiting example, a LAMP master mix will typically comprise 4 or 6 primers as described herein, together with a strand-displacing polymerase. When RNA is to be amplified (whether by PCR, LAMP or another technique) the master mix will typically comprise a reverse transcriptase.

A method of the invention may comprise one or more additional steps. Such additional steps may typically prepare a sample for nucleic acid amplification according to the methods of the invention. Standard techniques for sample preparation are known in the art, such as cell pelleting, cell lysis, enzymatic pre-digestion and/or RNA or DNA extraction, and could be readily incorporated into a method of the invention by one of ordinary skill in the art. By way of non-limiting example, where a method of the invention is carried out to determine the level of intracellular DNA and/or RNA (whether total or integrated), a method of the invention may comprise a step of pelleting the cells in the sample and/or lysing the cells. A method of the invention may comprise a step of treating a cell lysate with an enzyme such as EcoRI (or any enzyme which does not create a nick within the target region for the primers and/or probes of the invention) to improve the sensitivity of the method. Where a method of the invention is used to detect and/or quantify RNA, then a method of the invention may comprise an RNA extraction step prior to amplification (e.g. step (a)). In preferred embodiments when a crude cell lysate is used as the sample material, the combination of lysis and enzymatic digestion reduce the DNA viscosity and allow for further improvements in the reproducibility and sensitivity of the data obtained. A particularly preferred lysis and digestion protocol comprises or consists of contacting the lysate with a lysis buffer (such as the preferred lysis buffer described herein) and vortexing for between about 5 to about 30 seconds (preferably about 10-15 seconds), followed by incubation for between about 2-4 (e.g. 3) hours at 50°C-60°C (e.g. 55°C).

As discussed herein a method of the invention may be used is detect and/or quantify one or more additional target regions of target viral genome. Thus, a method of the invention may comprise one or more additional step to facilitate the detection and/or quantification of the one or more additional target region. To reduce DNA viscosity and break-up clumps, the following protocol may preferably be used: 65°C for 1 min, 96°C for 2 min, 65°C for 4 min, 96°C for 1 min, 65°C for 1 min, 96°C for 30 sec. A final step of incubating lysates at 95°C for 15 minutes may be carried out to ensure complete inactivation of the proteinase K.

For a one round amplification for RNA and/or DNA targets (such as that exemplified in Figure 2b), a reaction volume of from about 5pl to about 50pl may be used. Preferably a reaction volume of from about 5pl to about 30pl is used, with reaction volumes of about 20pl being particularly preferred. These reaction volumes may be used in combination with maximum ramp rates and/or fast cycling, and in combination may provide a significant time and cost saving compared with conventional methods.

For a method comprising a two-round amplification (such as the semi-nested format described herein and exemplified in Figure 2c), the first round may use a reaction volume of from about 15pl to about 50pl may be used. Preferably a reaction volume of from about 15pl to about 30pl is used, with reaction volumes of about 20pl being particularly preferred. Independently, the second round may use a reaction volume of from about 0.5pl to about 50pl may be used. Preferably a reaction volume of from about 0.5pl to about 30pl is used, with reaction volumes of about 5 - 20pl being particularly preferred. These reaction volumes may be used in combination with maximum ramp rates and/or fast cycling, and in combination may provide a significant time and cost saving compared with conventional methods.

The different method formats of the present inventions may have different applications. By way of non-limiting example, methods comprising one round amplification for RNA and/or DNA targets (e.g. an RT-qPCR format) may be useful for viral load determination. By way of a further nonlimiting example, a cell-based DNA format method may be used to monitor residual levels of viral nucleic acids in treatment-suppressed individuals. The different formats may be used with different sample types (e.g. cell/cell lysate samples or fluid (e.g. plasma) samples) to detect and/or quantify virus in different applications. Applications of the methods and assays of the invention include treatment monitoring, viral load determination, as a proxy measure of the latent viral reservoir, disease progression and/or prognosis, as well as diagnosis.

The methods and assays of the invention have the potential to offer numerous advantages over conventional methods/assays for the detection and/or quantification of viruses. By way of nonlimiting example, the methods of the invention may allow for detection of early-stage viral infections (e.g. acute stage HIV-1 infection, such as Fiebig disease stage I, II, III or IV). The methods of the invention may allow for the detection of low concentrations of virus (exemplary lower limits of detection are described herein). The methods of the invention are typically more sensitive, specific, accurate and with greater positive predictive value and/or greater negative predictive value compared with existing methods. The methods of the invention may be simpler to carry out and/or cheaper than those in the art. By way of non-limiting example, methods of the invention which use a crude DNA lysate as a sample are typically cheaper than methods involving complex extraction procedures. In addition, crude DNA lysis can maximise assay sensitivity by preventing the unnecessary loss of precious sample through more complicated DNA extraction procedures. Methods comprising detection and/or quantification of intracellular virus may be particularly preferred for disease and/or treatment monitoring, especially in resource-limited settings.

Primers and Probes

The LTR regions of a viral genome are typically well-conserved with subtypes/strains of lentiviruses in general because the LTRs are not transcribed within host cells and so are subject to low immune pressure, which would lead to sequence variation. Therefore, amplifying lentiviral or other viral LTR sequences (or other viral repeats or conserved regions) is advantageous, as it facilitates the detection of different viral subtypes, and improves cross-subtype coverage.

However, in practice assay design based on amplification of viral LTRs, particularly of HIV-1 LTRs, has proven complex. Without being bound by theory, it is believed that this is because sequencing enzymes become error-prone and drop off at the end of genomes. As such, there is limited information available regarding the LTR sequences of viruses (e.g. HIV-1) compared with information regarding other, more central regions of the genome. This is the case even where curated compendium databases are available, such as for HIV-1. The present inventions have devised a new approach to screening viral genomes and designing suitable primer and probe sequences for viral LTR regions, and in particular have designed primer probe sequences for HIV-1 LTRs with good crosssubtype specificity, as described and exemplified herein. This represents a new paradigm for the design of primer and/or probe sequences for virus detection and/or amplification, and can be readily extrapolated to other viruses (using other viral nucleic acids), particularly retroviruses and lentiviruses.

In developing the present invention, the inventors carried out methods of detecting and quantifying HIV-1 using the most promising oligonucleotide sequences that had been reported in the literature as being broadly cross-subtype specific. However, these primers were only about to accurately quantify 7 out of 20 (35%) strains of HIV-1 (see the Examples). On investigation, the inventors surprisingly found that these prior art oligonucleotides exhibited several mismatches against the NCBI/LANL HIV-1 compendium database, although no mismatches were present in the general sequence alignments.

In addition, where mismatches were identified using the compendium database, the inventors identified that inclusion of a wobble or inosine base at these positions within one or more of the primers and/or probe further improved the sensitivity, specificity, positive predictive value, negative predictive value and/or accuracy of the methods of the invention.

In particular, the invention provides a method for designing a oligonucleotide (e.g. a primer or probe) for use in the detection and/or quantification of a virus with improved cross-subtype specificity, said method comprising or consisting of the steps of: (a) conducting an alignment of a sequence panel of a plurality of different strains or subtypes of said virus (e.g. compendium sequences) for the viral subtypes of interest (b) identifying conserved regions (e.g. within viral LTR regions) within the viral subtypes; and (c) designing oligonucleotides that comprise or consist of a sequence within the conserved region, or are complementary to said sequence, preferably a conserved region of at least about 10 nucleotides, , such as from about 10 nucleotides to about 30 nucleotides, preferably from about 15 to about 25 nucleotides, such as aboutl5 - 20 nucleotides.

Said method may further comprise an addition step, step (d) following step (c) which any mismatches in the designed sequence are replaced with a wobble base or inosine base. If shorter primer or probe sequences are to be designed (e.g. for isothermal amplification such as by LAMP or another method as disclosed herein), said method may optionally not comprise a step of mismatch repair (e.g. by wobble or inosine replacement). This may be the case because shorter conserved regions are less likely to contain mismatches. Alternatively or in addition, said method may comprise a step of conducting a BLAST analysis following design of the one or more primer and/or probe sequence to verify that the designed sequences are specific for the virus in question. In particular, if shorter primer or probe sequences are to be designed (e.g. for isothermal amplification such as by LAMP or another method as disclosed herein), then inclusion of a BLAST analysis is particularly preferred to ensure that the primer and/or probe has the desired specificity to the virus in question.

In the context of retroviruses/lentiviruses (e.g. HIV-1), the invention provides a method for designing a oligonucleotide (e.g. a primer or probe) for use in the detection and/or quantification of said virus with improved cross-subtype specificity, said method comprising or consisting of the steps of (a) conducting an alignment of a sequence panel of a plurality of different strains or subtypes of said virus (e.g. compendium sequences) for the viral subtypes of interest; (b) identifying conserved regions (e.g. LTR); and (c) designing oligonucleotides that comprise or consist of a sequence within the conserved region (e.g. LTR), or are complementary to said sequence, preferably a conserved region of at least about 10 nucleotides, such as from about 10 nucleotides to about 30 nucleotides, preferably from about 15 to about 25 nucleotides, such as aboutl5 - 20 nucleotides.

Said method may further comprise an addition step, step (d) following step (c) which any mismatches in the designed sequence are replaced with a wobble base or inosine base. Alternatively or in addition, said method may comprise a step of conducting a BLAST analysis following design of the one or more primer and/or probe sequence to verify that the designed sequences are specific for the virus in question.

Additional further modifications may be made to primer and/or probe sequences designed according to the invention. By way of non-limiting example: (i) where multiple primers and/or probes to the same conserved region are designed, these can be designed to minimise cross-reactivity between the primer and/or probes (to eliminate or reduce primer dimers); (ii) avoid hairpins or other secondary structures; (iii) optimise GC content; (iv) insert specific 5' and/or 3' anchor nucleotides; and/or (v) insert or remove specific nucleotides at specific positions.

The invention also provides primer and/or probe sequences designed using these methods, as well as the use of said primer and/or probe sequences in the detection and/or quantification of viruses.

The primers and probes of the invention, and combinations thereof may be used to detect and/or quantify low concentrations, particularly low concentrations, of viral nucleic acid, as described herein.

The invention therefor provides primers, primer pairs and probes which comprise at least one inosine base and/or at least one wobble base at polymorphic sites Typically a primer, primer pair or probe comprises an inosine base or wobble base at each position identified as a mismatch through alignment of the compendium sequences, as described herein. Typically selection of each wobble base or inosine is selected to ensure that the chosen inosine/wobble base pairs with the mismatched nucleotides at a given position across the polymorphisms. Selection of a suitable wobble basis is within the routine practice of one of ordinary skill in the art.

The primers, primer pairs and probes of the invention typically hybridise to conserved sequences across different strains or subtypes of a particular virus. By way of non-limiting example, each of the primers and probes of the invention may bind to any conserved region of a virus identified using the strategy outlined herein.

By way of non-limiting example, primers, primer pairs and probes of the invention for use in detecting and/or quantifying retroviruses/lentiviruses (e.g. HIV-1) may hybridise to the viral LTR. In particular, for HIV-1, primers, primer pairs and probes of the invention may bind to the U3, R or U5 region of the viral (e.g. HIV-1) (5' or 3', preferably 5') LTR sequence. By way of non-limiting example, a primer may bind to the U5 or R region of a viral (e.g. HIV-1) LTR sequence. By way of further nonlimiting example, a probe may bind to the U5 or R region of a viral (e.g. HIV-1) (5' or 3', preferably 5') LTR sequence. The primers and probes may bind to different regions of a viral (e.g. HIV-1) (5' or 3', preferably 5') LTR sequence. By way of non-limiting example, in the context of a retrovirus/lentivirus (e.g. HIV-1) a primer may bind to the R region, and a probe may bind to the U5 region.

The primers and/or probes of the present invention may be designed for specific amplification techniques. For example, primers for PCR (e.g. qPCR) may have different constraints compared with primers for LAMP or other isothermal amplification techniques. Once a desired target (conserved) region has been identified according to the invention, suitable modifications to the base primer and/or probe sequences may be made using standard methodology to comply with the specific constrains of the desired amplification technique.

In some preferred embodiments which use PCR (particularly qPCR for amplification) primers designed according to the present invention may: (i) be from about 18 to about 30 nucleotides in length; (ii) have a T_m of between about 60-62°C; (iii) not comprise runs of 4 or more G nucleotides; and/or (iv) have a G/C content of from about 35 to about 65% (particularly 50%), particularly all of (i)- (iii). Primer pairs may preferably have T_m within ± about 2°C of each other.

In some preferred embodiments which use PCR (particularly qPCR for amplification) probes designed according to the present invention may: (i) be up to 30 nucleotides in length, such as from about 15 to about 30 nucleotides in length; (ii) not comprise any consecutive G nucleotides; (iii) have a G/C content of from about 40 to about 60%; (iv) not have a G as the 5' nucleotide; (v) have a T_m that is from about 4°C to about 6°C higher than the T_m of the primers. Probes may be designed to either the sense or antisense strand of the target nucleic acid.

In some preferred embodiments, which use PCR (particularly qPCR for amplification) the amplicon (nucleic acid sequence to be amplified) is between about 50 to about 150 nucleotides. Optimal target region length may vary depending on the specific amplification strategy for the virus in question and region of interest.

For embodiments in which virus is detected and/or quantified using LAMP to amplify viral nucleic acid, four to six primers to one or more identified conserved nucleic acid region are typically used. LAMP primers typically comprise a forward internal primer (FIP typically comprising the Flc and F2 regions), forward outer primer (F3), backward internal primer (BIP typically comprising Bic and B2 regions) and backward outer primer (B3). Optionally a forward loop primer (LF) and/or backward loop primer (LB) may also be used. The length of each primer or primer region may be independently selected from be up to 30 nucleotides in length, such as from about 15 to about 30 nucleotides in length, particularly from about 18 nucleotides to about 25 nucleotides in length. The T_m for each primer or primer region may be independently selected from about 57°C to about 67°C. For example: (i) for the Flc and Bic regions, a T_m of from about 64°C to about 66°C (e.g. about 65°C) may be selected; (ii) for the F2, F3, B2 and/or B3 regions from about 59°C to about 61°C (e.g. about 60°C); and/or (iii) for the loop primers from about 64°C to about 66°C (e.g. about 65°C). The 3' ends of the F2/B2, F3/B3, and LF/LB and the 5' end of Flc/Blc may be designed so that the free energy is -4 kcal/ mol or less. The G/C content of each primer or primer region may be independently selected from about 40 to about 60%, typically from about 50% to about 60%. Each primer, particularly the internal (forward and/or backward) primer(s) may be designed such that they do not form secondary structures. The 3' ends of the LAMP primers may be designed such that they are not complementary, in order to prevent the formation of primer dimers. Each of these constraints may be selected independently or in any combination. Software (e.g. PimerExplorer at https://primerexplorer.j /e/) is available in the art to assist with LAMP primer design, once a target (conserved) region has been identified according to the present invention.

In some preferred embodiments, which use LAMP, the amplicon less than 250 nucleotides, typically is between about 50 to about 150 nucleotides. Optimal target region length may vary depending on the specific amplification strategy for the virus in question and region of interest.

In methods (e.g. PCR-based methods) which use a first and second primers which hybridise to different regions of the viral (e.g. HIV-1) LTR sequence, preferably said first and second primers hybridise to different regions of the 5' LTR sequence. Preferably said first primer hybridises to the R region of the 5' LTR sequence. Alternatively or in addition, preferably said second primer hybridises to the U5 region of the 5' LTR sequence.

The invention provides a primer that hybridises to a nucleic acid sequence within a target region of bases 500 to 550 of the LTR (e.g. HIV-1 LTR), particularly a nucleic acid sequence within a target region of bases 520 to 545 of the LTR (e.g. HIV-1 LTR), more particularly a nucleic acid sequence with a target region of bases 525 to 543 of the LTR (e.g. HIV-1 LTR). The invention provides a primer that comprises or consists of the sequence ATGCCACGTAAGCGAAACTTCAATAAAGCTTGCCTTGA (SEQ ID NO: 1), or a variant differing by up to five (particularly one or two) nucleotides from SEQ. ID NO: 1. The invention also provides a primer that comprises or consists of the sequence TCAATAAAGCTTGCCTTGA (SEQ ID NO 3), or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 3.

The invention also provides a primer that hybridises to a nucleic acid sequence within a target region of bases 550 to 600 of the LTR (e.g. HIV-1 LTR), particularly a nucleic acid sequence within a target region of bases 575 to 600 of the LTR (e.g. HIV-1 LTR), more particularly a nucleic acid sequence with a target region of bases 582 to 599 of the LTR (e.g. HIV-1 LTR). The invention provides a primer that comprises or consists of the sequence AGGGATCTCTAGITACCA(SEQ ID NO: 2), or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 2.

In methods (e.g. PCR-based methods) which use first and second primers which hybridise to different regions of the viral (e.g. HIV-1) LTR sequence, preferably said first primer hybridises to a nucleic acid sequence within a target region of bases 500 to 550 of the LTR (e.g. HIV-1 LTR), particularly a nucleic acid sequence within a target region of bases 520 to 545 of the LTR (e.g. HIV-1 LTR), more particularly a nucleic acid sequence with a target region of bases 525 to 543 of the LTR (e.g. HIV-1 LTR). Accordingly, said first primer may comprise or consist of the sequence SEQ ID NO: 1, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 1.

Alternatively or in addition, in methods which use first and second primers which hybridise to different regions of the viral (e.g. HIV-1) LTR sequence, preferably said second primer hybridises to a nucleic acid sequence within a target region of bases 550 to 600 of the LTR (e.g. HIV-1 LTR), particularly a nucleic acid sequence within a target region of bases 575 to 600 of the LTR (e.g. HIV-1 LTR), more particularly a nucleic acid sequence with a target region of bases 582 to 599 of the LTR (e.g. HIV-1 LTR). Accordingly, said second primer may comprise or consist of the sequence SEQ ID NO: 2, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 2.

In methods which use a primer for an Alu sequence and a first primer for a viral LTR sequence, preferably said first primer for a viral LTR sequence hybridises to a nucleic acid sequence within a highly conserved target region of the LTR as identified according to the present invention. Said primer for a viral LTR sequence may hybridise to the R region of the 5' or 3' (preferably 5') LTR sequence. A second primer for a viral LTR sequence may hybridise to the U5 region of the 5' or 3' (preferably 5') LTR sequence. In particular, in methods which use a primer for an Alu sequence and a primer for a HIV-1 LTR sequence, preferably said primer for a HIV-1 LTR sequence hybridises to a nucleic acid sequence within bases 500 to 600 of the HIV-1 LTR, such as a nucleic acid sequence within a target region of bases 500 to 550 of the HIV-1 LTR, particularly a nucleic acid sequence within a target region of bases 520 to 545 of the HIV-1 LTR, more particularly a nucleic acid sequence with a target region of bases 525 to 543 of the HIV-1 LTR. Accordingly, said primer for a HIV-1 LTR sequence may comprise or consist of the nucleic acid sequence selected from SEQ ID NO: 1, or a variant differing by up to five (particularly one or two) nucleic acids from SEQ. ID NO: 1 and SEQ ID NO 3, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 3. Said second primer for a HIV-1 LTR sequence typically hybridises to a nucleic acid sequence within a target region of bases 575 to 600 of the HIV-1 LTR, more particularly a nucleic acid sequence with a target region of bases 582 to 599 of the HIV-1 LTR. Accordingly, said primer for a HIV-1 LTR sequence may comprise or consist of the sequence SEQ ID NO: 2, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 2. By way of non-limiting example, an Alu primer may comprise or consist of the Alul primer of SEQ ID NO: 25, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 25. By way of a further non-limiting example, an Alu primer may comprise or consist of the Alu2 primer of SEQ ID NO: 26, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 26.

In methods which comprise or consist of one round amplification of DNA/RNA (e.g. a nonnested PCR method as described herein), a first (forward) primer may hybridise to a first nucleic acid sequence within a highly conserved target region, such as an LTR. In the context of HIV-1, a first (forward) primer may hybridise to a nucleic acid sequence of bases 500 to 600 of the HIV-1 LTR , particularly a nucleic acid sequence within a target region of bases 520 to 545 of the LTR (e.g. HIV-1 LTR), more particularly a nucleic acid sequence with a target region of bases 525 to 543 of the LTR (e.g. HIV-1 LTR). Accordingly, said first primer may comprise or consist of the sequence of SEQ ID NO 3, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 3

Alternatively or in addition, in methods which comprise or consist of one round amplification of DNA/RNA, a second (reverse) primer may hybrids to a second nucleic acid sequence within a highly conserved target region of, such as an LTR. In the context of HIV-1, a second (reverse) primer may hybridise to a nucleic acid sequence of bases 525 to 600 of the HIV-1 LTR, particularly a nucleic acid sequence within a target region of bases 575 to 600 of the HIV-1 LTR, more particularly a nucleic acid sequence with a target region of bases 582 to 599 of the HIV-1 LTR. Accordingly, said second primer may comprise or consist of the sequence SEQ ID NO: 2, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 2.

Newer amplification methods can work with shorter primers and without a probe region. In addition or alternatively, primers for use with different techniques, particularly isothermal techniques such as LAMP may require different or multiple target sequences within conserved regions identified according to the invention. One of ordinary skill in the art will be able to devise suitable primer sequences and primer sets as a matter of routine once suitable target regions have been identified. By way of non-limiting example, nucleotides 500 to 550 of the HIV-1 LTR, particularly nucleotides 520 to 545 of the HIV-1 LTR, more particularly nucleotides 525 to 543 of the HIV-1 LTR, even more particularly a nucleic acid sequence comprising or consisting of SEQ ID NO: 3, or a variant differing by up to five (particularly one or two) nucleotides from SEQ. ID NO: 3 may be used as an F2 region for LAMP. By way of further non-limiting example, alternatively or in addition, nucleotides 550 to 600 of the HIV-1 LTR, particularly nucleotides 575 to 600 of the HIV-1 LTR, more particularly nucleotides 582 to 599 of the HIV-1 LTR, even more particularly a nucleic acid sequence comprising or consisting of SEQ ID NO: 2, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 2 may be used as an Fl region for LAMP. The region downstream of the HIV-1 LTR may be used as an F3 region for LAMP. By way of further non-limiting example, alternatively or in addition, nucleotides 480 to 530 of the HIV-1 LTR, more particularly nucleotides 496 to 516 of the HIV-1 LTR, even more particularly a nucleic acid sequence comprising or consisting of SEQ ID NO: 12 SEQ ID NO: 12, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 12 may be used as an F3 region for LAMP. The Bl and B2 regions for LAMP may each independently be selected a nucleotides 630 to 710 (that spans into the pre-GAG region of HIV-1).

Specific examples of primers that may be used for LAMP of HIV-1 include one or more primer which comprises, consists or is complementary to a sequence selected from:

• a nucleic acid sequence of bases 490 to 520 of HIV-1 LTR, particularly bases 495 to 518, more particularly bases 496 to 516, such as a primer that comprises or consists of the sequence of SEQ ID NO: 12, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 12, which may optionally be used as an F3 or F2 LAMP primer region;

• a nucleic acid sequence of bases 520 to 550 of HIV-1 LTR, particularly bases 521 to 548, more particularly bases 522 to 546, such as a primer that comprises or consists of the sequence of SEQ ID NO: 13, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 13, which may optionally be used as an F2 or Fl LAMP primer region;

• a nucleic acid sequence of bases 545 to 575 of HIV-1 LTR, particularly bases 547 to 573, more particularly bases 549 to 572, such as a primer that comprises or consists of the sequence of SEQ ID NO: 14, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 14, which may optionally be used as an Fl LAMP primer region;

• a nucleic acid sequence of bases 580 to 600 of HIV-1 LTR, particularly bases 581 to 600, more particularly bases 582 to 599, such as a primer that comprises or consists of the sequence of SEQ ID NO: 15, or a variant differing by up to five (particularly one or two) nucleotides from SEQ. ID NO: 15, which may optionally be used as an Fl LAMP primer region;

• a nucleic acid sequence of bases 595 to 625 of HIV-1 LTR, particularly bases 598 to 623, more particularly bases 599 to 622, such as a primer that comprises or consists of the sequence of SEQ ID NO: 16, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 16, which may optionally be used as an Fl or B2 LAMP primer region;

• a nucleic acid sequence of bases 630 to 655 of HIV-1 in the pre-GAG region (633-789bp of the HIV-1 genome), particularly bases 632 to 654, more particularly bases 633 to 652, such as a primer that comprises or consists of the sequence of SEQ ID NO: 17, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 17, which may optionally be used as a Bl LAMP primer region;

• a nucleic acid sequence of bases 605 to 635 of HIV-1 LTR, particularly bases 607 to 634, more particularly bases 609 to 633, such as a primer that comprises or consists of the sequence of SEQ ID NO: 18, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 18, which may optionally be used as a Bl LAMP primer region;

• a nucleic acid sequence of bases 650 to 685 of HIV-1 pre-GAG, particularly bases 651 to 682, more particularly bases 652 to 680, such as a primer that comprises or consists of the sequence of SEQ ID NO: 19, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 19 , which may optionally be used as a B2 or B3 LAMP primer region; and/or

• a nucleic acid sequence of bases 675 to 705 of HIV-1 pre-GAG, particularly bases 677 to 700, more particularly bases 680 to 699, such as a primer that comprises or consists of the sequence of SEQ ID NO: 20, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 20, which may optionally be used as a B3 LAMP primer region;

• or any combination thereof.

As described herein, primers for isothermal amplification, such as LAMP primers, and particularly the above-exemplified LAMP primers may be modified, for example by: (i) addition of a tag or other detectable means; and/or (ii) incorporating one or more inosine or wobble base where there are mismatches between the conserved regions in different viral subtypes or strains. In addition, as primers for isothermal amplification techniques such as LAMP may be shorter than those used for PCR, then any of the above regions/nucleic acids/primers may be shortened by up to 5 nucleotides, such as by any of 1, 2, 3, 4 or 5 nucleotides. The invention also provides one or more oligonucleotide probes suitable for use in a method of the invention. Said probe typically specifically hybridises with the amplified viral nucleic acid and can be detected, thus facilitating the detection and/or quantification of the virus. A detectable probe means an oligonucleotide that is capable of emitting a signal, either directly or indirectly, through the use of various labels, preferably only when hybridised to its target sequence.

A probe can be labelled by any technique well known to those skilled in the art. The probe preferably carries a fluorescent moiety, i.e. chemicals which fluoresce when exposed to ultraviolet light. A number of fluorescent materials are known and be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA Blue, and Lucifer Yellow.

For methods comprising isothermal amplification, one or more of the primers may be labelled with a tag or other detection means, and so may also be considered/described as a probe. By way of non-limiting example, one or more of the primers may be labelled with a detection means that gives a colorimetric readout (i.e. in the visible range of the light spectrum). Such colorimetric detection means may allow signal detection using a spectrophotometer or photographically, even potentially via an smart phone camera. This could allow the use of a smart phone app to detect and quantify the signal. The method can be adapted to these amplification techniques such as LAMP,CRISPR-based amplification, NASBA, SDA and/or RPA techniques.

Thus, a probe of the invention may comprise a detectable moiety, such as a fluorescent moiety, colorimetric label, enzymatic label, or radiolabel, preferably a colorimetric label or fluorescent moiety (also referred to as a fluorescent dye). Examples of radiolabels include ³H, ¹⁴C, ³²P and ³⁵S. Enzyme labels can be detected by any of the known colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme may be conjugated to the probe by reaction with bridging molecules such as carbodiimides, diisocyanates, or glutaraldehyde.

A probe of the invention may comprise a fluorescent dye and one or two quencher dye(s), particularly one or two non-fluorescent quenchers (NFQ). A fluorescent dye may be present at the 5' or 3' end of the probe, preferably at the 5' end. An NFQ. may be present at the 3' or 5' end of the probe, preferably the 3' end. Examples of suitable dyes are known in the art and can be readily selected by the skilled person. Non-limiting examples of fluorescent dyes include 6- carboxyfluorescein (6-FAM or FAM), 2'-chloro-7'phenyl-l,4-dichloro-6-carboxy-fluorescein (VIC), hexachloro-fluorescein (HEX), tetrachloro fluorescein (TET), or NED. Examples of quenchers include TAMRA and Atto quenchers such as Atto540Q, Atto575Q and Atto612Q. NFQ include black hole quenchers (BHQ) such as BHQ-1, BHQ-2, Dabcyl, malachite green, QSY quenchers such as QSY7, QSY9, QSY21 and QSY35, Qxl quenchers, Iowa black FQ, Iowa black RQ, and IRDye QC-1. A probe of the invention may comprise a minor groove binder (MGB) moiety. A MGB may be present at the 3' or 5' end of the probe, preferably the 3' end. Typically inclusion of MGB moiety increases the melting temperature (T_m) of the probe and stabilises probe/target hybrids. In some embodiments a probe of the invention comprises a 3' MGB-NFQ combination. In some embodiments, a probe of the invention comprises a 5' fluorescent dye (e.g. a FAM dye such as 6FAM) and a 3' MGB-NFQ. combination. Other non-limiting examples of dye/quencher combinations include FAM and ZEN/lowa Black™FQ, SUN™ and ZEN/lowa Black™FQ, HEX and ZEN/lowa Black™FQ, Cy3 and Iowa Black RQ, Texas Red-X and Iowa Black RQ, and Cy5 and TAO/lowa Black RQ. A probe of the invention may comprise one or more modified base, such as a locked nucleic acid (LNA) to improve assay sensitivity, structural stability and/or T_m. Other possible probe structures include dual-labelled probes (a quencher dye at one end and a fluorescent moiety at the other) with self-complimentary ends that for a quenched hairpin structure in the absence of the target nucleic acid sequence.

The invention also provides a probe that hybridises to a nucleic acid sequence within a target conserved region identified according to the invention, such as a nucleic acid region with a viral LTR region (particularly in the context of retroviral and/or lentiviral detection/quantification). By way of non-limiting example, specifically in relation to HIV-1, the invention provides a probe that hybridises to a nucleic acid sequence within a target region of bases 540 to 580 of the LTR (e.g. HIV-1 LTR), particularly a nucleic acid sequence within a target region of bases 550 to 575 of the LTR (e.g. HIV-1 LTR), more particularly a nucleic acid sequence with a target region of bases 552 to 574 of the LTR (e.g. HIV-1 LTR). The invention also provides a probe that comprises or consists of the sequence of ACAGAYGGGCACACACIACT (SEQ ID NO: 5), or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 5. A probe of the invention may comprise or consist of the sequence SEQ ID NO: 5, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 5 and a 5' fluorescent moiety. A probe of the invention may comprise or consist of the sequence SEQ ID NO: 5, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 5, a 5' fluorescent moiety and a 3' quencher, such as a 3' NFQ. A probe of the invention may comprise or consist of the sequence SEQ ID NO: 5, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 5, a 5' fluorescent moiety and a 3' quencher, such as a 3' NFQ and a 3' MGB. In some embodiments a probe of the invention may comprise the sequence SEQ ID NO: 5, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 5, a 5' FAM dye and a 3' quencher, such as a 3' NFQ and a 3' MGB. In some embodiments, a probe of the invention comprises or consists of 5'6-FAM/ACAGAYGGGCACACACIACT/MGBNFQ-3'.

Methods of the invention may use several different probes, e.g. two probes that specifically hybridise to the amplified viral nucleic acid, and that may be labelled by labels of different types. The invention also provides a set of oligonucleotides useful in carrying out a method of the invention. Said set of oligonucleotides may comprise or consist of any of the oligonucleotides described herein, in any combination. Said set of oligonucleotides may comprise or consist of (i) one or more forward primer; (i) a reverse primer; and/or (iii) a probe. For methods involving one-round application, said set of oligonucleotides may comprise or consist of (i) one forward primer; (i) a reverse primer; and/or (iii) a probe. For methods involving two-rounds of application (e.g. the semi-nested methods described herein), said set of oligonucleotides may comprise or consist of (i) two or more forward primers; (i) a reverse primer; and/or (iii) a probe. Said set of oligonucleotides may comprise one or more LAMP primer, such as any combination of the LAMP primer sequences described herein, or one or more LAMP primers comprising one or more of the LAMP primer regions described herein.

In some preferred embodiments, the invention provides a set of oligonucleotides comprising or consisting of: (i) SEQ ID NO 3, or a variant differing by up to five (particularly one or two) nucleotides from SEQ. ID NO: 3; (ii) SEQ ID NO 2, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 2; and (iii) SEQ ID NO: 5, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 5, preferably 5'6- FAM/ACAGAYGGGCACACACIACT/MGBNFQ-3'. Said set of oligonucleotides may be preferred for methods involving one-round application.

In some preferred embodiments, the invention provides a set of oligonucleotides comprising or consisting of: (i) SEQ ID NO: 1, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 1 and SEQ ID NO: 4, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 4; ii) SEQ ID NO 2, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 2; and (iii) SEQ ID NO: 5, or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 5, preferably 5'6- FAM/ACAGAYGGGCACACACIACT/MGBNFQ-3'. Said set of oligonucleotides may be preferred for methods involving two-rounds of application (e.g. the semi-nested methods described herein)

Tags

The tag sequence is a nucleic acid sequence selected so that it is not capable of hybridising to the viral genome nor to the cellular (e.g. human) genome, especially under the conditions of stringency used for the step (a) of the method of the invention.

The tag sequence may be any suitable length, such as between 15 and 25 nucleotides in length. The tag sequence may comprise or consist of any sequence, provided that said tag sequence is completely foreign to the host and target organism. Byway of non-limiting example, the tag sequence may comprise or consist of a sequence from the genome of a Lambda phage, such as the lambda phage SEQ ID NO: 4 or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 4. The primer for the tag sequence may also comprise or consist of SEQ ID NO: 4 or a variant differing by up to five (particularly one or two) nucleotides from SEQ ID NO: 4.

Without being bound by theory, it is believed that the use of a "tag" on one of the primers (e.g. in a two-round method such as the preferred semi-nested PCR methods of the invention) may allow for amplifications across regions of incomplete homology increasing the chance of good cross-subtype coverage. This is particularly believed to be the case when a tag is used in combination with less stringent initial annealing temperatures (such as the "step-up" cycling parameters described herein). Without being bound by theory, it is believed that use of a tag allows subsequent amplification of only specific amplicons from the first round by reducing non-specific amplification of any primer-dimers or nonspecific artefacts generated in the primary PCR thus maintaining PCR specificity.

The methods and assays of the invention may be multiplex methods/assays. By "multiplex", it is meant that one or more additional target regions of the viral genome is detected and/or quantified within the same reaction. In the case of retroviral or lentiviral vectors such as HIV, particularly HIV-1 said one or more additional target region mays be selected from highly conserved regions such as the integrase gene and/or the polymerase gene.

The more conserved region of the polymerase gene runs from 2445 - 5096 bp (e.g. of the reference HIV-1 genome SEQ ID NO: 23). The integrase gene is located within this region (4529 to 5096bp (± 10 nucleotides, preferably ± 5 nucleotides)), SEQ ID NO: 22. The invention may comprise the use of one or more primer or probe sequence comprising or complementary to a sequence within this region. Non-limiting examples of this nucleotides within this conserved region that may be used to generate primer and/or probe sequences of the invention include nucleotides 4746 - 4768 (± 10 nucleotides, preferably ± 5 nucleotides) for a forward primer, nucleotides 4956 to 4976 (± 10 nucleotides, preferably ± 5 nucleotides) for a reverse primer and/or nucleotides 4899 to 4922(± 10 nucleotides, preferably ± 5 nucleotides) for a probe. In some embodiments, one or more primer or probe sequence may comprise or be complementary to a sequence within nucleotides 4746 to 4922 (± 10 nucleotides, preferably ± 5 nucleotides).

A multiplex method/assay of the invention comprise the detection and/or quantification of a reference gene (e.g. RNaseP or HPRT). The inclusion of a reference gene allows the amount of virus per cell to be quantified using the same sample, and so is particularly suited for samples which may comprise limited nucleic acid material (e.g. crude lysates). Thus, the inclusion of a reference gene for quality control and/or quantification with respect to overall background mRNA amplification and/or with respect to cell numbers within the same sample. Thus, the inclusion of a reference gene improves data accuracy and/or quality and makes better use of limited samples.

Alternatively, the methods and assays of the invention may be used in combination with other methods/assays targeting one or more additional target region or gene, such as a viral integrase gene and/or a viral polymerase gene. When used in combination with other methods and/or assays, these may be carried out simultaneously or sequentially (in series or in parallel).

Such multiplex assays, particularly when they allow for quantification of peripheral blood nucleic acid, detect nucleic acids even when the plasma viral load is undetectable as measured by current commercial tests. These assay formats will thus be more sensitive than current viral load assay and potentially improve on them.

The invention therefore provides methods, particularly methods which use crude cell lysates and lyophilised reagents, that can be used for treatment monitoring, especially in POC devices in resource-limited settings.

Kits and Master Mixes

The invention provides a kit for performing a method or assay of the invention, said kit comprising or consisting of: one or more primer or probe of the invention, or a set of oligonucleotides of the invention, and optionally one or more additional reagent for carrying out said method or assay. A kit of the invention may also comprise instructions for carrying out a method or assay of the invention.

A kit of the invention may comprise or consist of: (i) a first container containing a first and second primer for a viral LTR sequence and a second container containing the second primer for a viral LTR sequence and a primer for the tag sequence; and (ii) a detectable probe, wherein the detectable probe may be contained in same container as the second primer and/or probe, or in a separate container.

A kit of the invention may comprise or consist of: (i) a first container containing a first primer for a viral LTR sequence, a second container containing a second primer for a viral LTR sequence, and a third container containing a primer for the tag sequence; and (ii) a detectable probe, wherein the detectable probe may be contained in same container as the second primer and/or probe, or in a separate container.

A kit of the invention may comprise or consist of: (i) a first container containing an Alu primer and a primer for a viral LTR sequence and a second container containing the primer for a viral LTR sequence and a primer for the tag sequence; and (ii) a detectable probe, wherein the detectable probe may be contained in same container as the primer for a viral LTR sequence and/or probe, or in a separate container.

A kit of the invention may comprise or consist of: (i) a first container containing an Alu primer, a second container containing a primer for a viral LTR sequence, and a third container containing a primer for the tag sequence; and (ii) a detectable probe, wherein the detectable probe may be contained in same container as the primer for a viral LTR sequence and/or probe, or in a separate container.

Optionally said kit may comprise one or more additional reagent for carrying out said method. Any appropriate thermocyclers and master mixes may be used in a method of the invention. The invention provides an ambient temperature viral quantification kit, particularly an ambient temperature HIV-1 DNA and/or RNA quantification kit. Such ambient temperature kits are particularly useful in resource-limited settings that cannot maintain a cold-chain or perform complicated DNA/RNA extraction procedures. Such kits (and assays/methods conducted using said kits) allow for the detection and/or quantification of spikes in viral nucleic acid (e.g. HIV-1 DNA) during treatment. As such, these kits/methods/assays may be used to indicate the potential emergence of drugresistance and viral escape, and suggest further analysis (e.g. sequencing) to detect specific mutations and/or suggest an alternative drug regimen. As such, the kits/methods/assays may be used in combination with additional methods or techniques such as sequencing, typically as a precursor to such additional methods/techniques. An ambient temperature viral quantification kit, particularly an ambient temperature HIV-1 DNA and/or RNA quantification kit, may comprise lyophilised reagents (such as described herein), and/or may comprise liquid reagents that do not required cold storage. One or more, preferably all of the components of a kit of the invention may be lyophilised or a liquid that does not require cold storage. In embodiments where a kit of the invention comprise one or more reagent which is not amenable to lyophilisation (particularly fluorescent dyes such as ROX), the one or more reagent that is not amenable to lyophilisation (e.g. ROX) will be present in the kit in a separate container or vial, preferably in a liquid form that does not require cold storage, and the remaining reagents will be present in lyophilised form. Such lyophilised and ambient temperature kits are p53particularly amenable to resource constrained settings.

In addition to a suitable master mix, such as those described herein, a kit of the invention may comprise separate vials of one or more of: (i) a fluorescent dye (e.g. ROX); (ii) a lyophilised lysis buffer; (iii) a synthetic template for HIV and/or RNaseP; or any combination thereof, and optionally: (a) a high, medium and/or low (preferably all three) calibration standard; and/or (b) molecular grade water to reconstitute the lyophilised ingredients. Equivalents to this kit composition would be within the routine grasp of one of ordinary skill in the art. For example, a kit may comprise a first separate vial of lyophilised lysis buffer and a second separate vial of lyophilised proteinase K, rather than the proteinase being comprised in the lyophilised lysis buffer.

Any suitable lysis buffer may be provided, particularly if a kit is suitable or intended for use with lysate samples as described herein. Suitable lysis buffers are available commercially. In some embodiments, a lysis buffer may comprise or consist of lOmM Tris HCL pH8 (Ambion), 50mM KCI (Ambion) and 0.4mg/ml Proteinase K (Qiagen) in molecular biology grade water. This lysis buffer may be lyophilised for including in a kit of the invention, either as a single lyophilised product, or with the proteinase K lyophilised separately to the other lysis buffer components.

As exemplified herein, carrier RNA and/or DNA, particularly tRNA (e.g. yeast tRNA) may be added to PCR master-mixes and sample diluents to improve assay/method efficiency, quantification sensitivity and precision at low viral levels (such as those found in treated subjects). By way of nonlimiting example, each PCR reaction in a method of the invention may comprises a concentration of lOpg/mL of tRNA (particularly yeast tRNA). Optionally this may be achieved by using a master-mix which comprises a concentration of lOpg/mL of tRNA (particularly yeast tRNA), as described herein.

A one round amplification master mix may comprise any suitable reagent(s), examples of which are readily (commercially) available, such as the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems). Other master mixes or master mix reagents, including other commercial master mixes which provides comparable assay performance may also be used.

A master mix may comprise a first (forward) primer and a second (reverse) primer, optionally further comprising or consisting of (i) a carrier DNA or RNA (e.g. tRNA, particularly yeast tRNA) and/or (ii) a probe. By way of non-limiting example, a master mix may comprise 250nm-lpM (particularly 500-750nM) of a first (forward) primer, 250nm-lpM (particularly 500-750nM) of a second (reverse) primer, and optionally: (i) 50nm-500nM (particularly 150-250nM) of a probe and/or (ii) l-50ng/mL (particularly 5-15ng/mL) of (yeast) tRNA.

For a one round amplification for RNA and/or DNA targets (such as that exemplified in Figure 2b), a master mix may comprise or consist of the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems) with a first (forward) primer and a second (reverse) primer, optionally further comprising or consisting of (i) a carrier DNA or RNA (e.g. tRNA, particularly yeast tRNA) and/or (ii) a probe. By way of nonlimiting example, a master mix may comprise or consist of the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems) with250nm-lpM (particularly 500-750nM) of a first (forward) primer, 250nm-lpM (particularly 500-750nM) of a second (reverse) primer, and optionally: (i) 50nm-500nM (particularly 150-250nM) of a probe and/or (ii) l-50ng/mL (particularly 5-15ng/mL) of (yeast) tRNA. Other master mixes or master mix reagents, including other commercial master mixes which provides comparable assay performance may be substituted for the qPCRBIO Probe Mix Lo-Rox reagents. The amount of target nucleic acid to be added to the master mix may be readily determined and selected by a skilled person based on the tolerance of the specific master mix or reagent systems (e.g. primers and/or probe). By way of non-limiting example, 1X10⁶-1X10⁷, such as l-7xl0⁶, particularly l-4xl0⁶ input copies of target nucleic acid may be added to the master mix.

A preferred one round amplification master mix may comprise or consist of the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems) with 600 nM of a first (forward) primer, 600 nM of a second (reverse) primer, 200 nM of a probe and 10 ng/ml yeast tRNA. Up to 3x10^s input-copies of target nucleic acid (e.g. enzyme-digested target HIV DNA) may be added to this specific master mix but larger amounts may be accommodated by other master mixes and /or if the reaction volume is increased above 20pl.

For a method comprising two round amplification (such as the semi-nested format described herein and exemplified in Figure 2c), the first (and/or second) round master mix may comprise any suitable reagent(s), examples of which are readily (commercially) available, such as the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems). Other master mixes or master mix reagents, including other commercial master mixes which provides comparable assay performance may also be used.

For a method comprising two round amplification (such as the semi-nested format described herein and exemplified in Figure 2c), a first round master mix may comprise a first (forward) primer and a second (reverse) primer, optionally further comprising or consisting of (i) a carrier DNA or RNA (e.g. tRNA, particularly yeast tRNA) and/or (ii) a probe. By way of non-limiting example, a first round master mix may comprise 250nm-lpM (particularly 500-750nM) of a first (forward) primer, 250nm- lpM (particularly 500-750nM) of a second (reverse) primer, and optionally: (i) 50nm-500nM (particularly 150-250nM) of a probe and/or (ii) l-50ng/mL (particularly 5-15ng/mL) of (yeast) tRNA.

For a method comprising two round amplification (such as the semi-nested format described herein and exemplified in Figure 2c), a first round master mix may comprise or consist of the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems) with a first (forward) primer and a second (reverse) primer, optionally further comprising or consisting of (i) a carrier DNA or RNA (e.g. tRNA, particularly yeast tRNA) and/or (ii) a probe. By way of non-limiting example, a first round master mix may comprise or consist of the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems) with250nm-lpM (particularly 500-750nM) of a first (forward) primer, 250nm-lpM (particularly 500-750nM) of a second (reverse) primer, and optionally: (i) 50nm-500nM (particularly 150-250nM) of a probe and/or (ii) 1- 50ng/mL (particularly 5-15ng/mL) of (yeast) tRNA. Other master mixes or master mix reagents, including other commercial master mixes which provides comparable assay performance may be substituted for the qPCRBIO Probe Mix Lo-Rox reagents. The amount of target nucleic acid to be added to the first round master mix may be readily determined and selected by a skilled person based on the tolerance of the specific master mix or reagent systems (e.g. primers and/or probe). By way of non-limiting example, 1X10⁶-1X10⁷, such as 1- 7x10^s, particularly l-4xl0⁶ input copies of target nucleic acid may be added to the master mix.

A preferred first round amplification master mix may comprise or consist of the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems) with any amount of a first (forward) primer, any amount of a second (reverse) primer, and an optimized range yeast tRNA (typically 2 to 15ng/ml) or other carrier RNA or nucleic acid species. Up to 3x10^s input-copies of target nucleic acid (e.g. enzyme- digested target HIV DNA) is recommended to be added to this master mix but larger amounts may be accommodated by other master mixes and /or if the reaction volume is increased above 20pl.

For a method comprising two round amplification (such as the semi-nested PCR format described herein and exemplified in Figure 2c), the second round master mix may comprise any suitable reagent(s), examples of which are readily (commercially) available, such as the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems). Other master mixes or master mix reagents, including other commercial master mixes which provides comparable assay performance may also be used.

For a method comprising two round amplification (such as the semi-nested PCR format described herein and exemplified in Figure 2c), a second round master mix may comprise the second (reverse) primer, and a tag primer, optionally further comprising or consisting of (i) a carrier DNA or RNA (e.g. tRNA, particularly yeast tRNA) and/or (ii) a probe. By way of non-limiting example, a second round master mix may comprise 250nm-lpM (particularly 500-750nM) of a first (forward) primer, 250nm-lpM (particularly 500-750nM) of a second (reverse) primer, and optionally: (i) 50nm-500nM (particularly 150-250nM) of a probe and/or (ii) l-50ng/mL (particularly 5-15ng/mL) of (yeast) tRNA.

For a method comprising two round amplification (such as the semi-nested format described herein and exemplified in Figure 2c), a first round master mix may comprise or consist of the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems) with the second (reverse) primer, and a tag primer, optionally further comprising or consisting of (i) a carrier DNA or RNA (e.g. tRNA, particularly yeast tRNA) and/or (ii) a probe. By way of non-limiting example, a second round master mix may comprise or consist of the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems) with 250nm-lpM (particularly 500-750nM) of the second (reverse) primer, 250nm-lpM (particularly 500-750nM) of a tag primer, and optionally: (i) 50nm-500nM (particularly 150-250nM) of a probe and/or (ii) l-50ng/mL (particularly 5-15ng/mL) of (yeast) tRNA. Other master mixes or master mix reagents, including other commercial master mixes which provides comparable assay performance may be substituted for the qPCRBIO Probe Mix Lo-Rox reagents.

O.lpl or more, such as 0.1-50 pl, particularly l-10pls of the amplification product from the first round may be added to the second round master mix. A preferred second round amplification master mix may comprise or consist of the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems) with 600 nM of the second (reverse) primer, 600 nM of a tag primer, 200 nM of a probe and 10 ng/ml yeast tRNA. 2pl of the amplification product from the first round may be added to the second round master mix.

For a method comprising two round amplification for the detection and/or quantification of an integrated virus (such as described herein), the first (and/or second) round master mix may comprise any suitable reagent(s), examples of which are readily (commercially) available, such as the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems). Other master mixes or master mix reagents, including other commercial master mixes which provides comparable assay performance may also be used.

For a method comprising two round amplification for the detection and/or quantification of an integrated virus (such as described herein), a first round master mix may comprise a primer for an Alu sequence and a first primer for a conserved viral sequence (e.g. LTR), optionally further comprising or consisting of (i) a carrier DNA or RNA (e.g. tRNA, particularly yeast tRNA) and/or (ii) a probe. By way of non-limiting example, a first round master mix may comprise 250nm-lpM (particularly 500- 750nM) of a primer for an Alu sequence, 250nm-lpM (particularly 500-750nM) of a first primer for a conserved viral sequence (e.g. LTR) and optionally: (i) 50nm-500nM (particularly 150-250nM) of a probe and/or (ii) l-50ng/mL (particularly 5-15ng/mL) of (yeast) tRNA.

For a method comprising two round amplification (such as described herein), a first round master mix may comprise or consist of the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems) with a primer for an Alu sequence and a first primer for a conserved viral sequence (e.g. LTR), optionally further comprising or consisting of (i) a carrier DNA or RNA (e.g. tRNA, particularly yeast tRNA) and/or (ii) a probe. By way of non-limiting example, a first round master mix may comprise or consist of the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems) with 250nm-lpM (particularly 500-750nM) of a primer for an Alu sequence, 250nm-lpM (particularly 500-750nM) of a first primer for a conserved viral sequence (e.g. LTR) and optionally: (i) 50nm-500nM (particularly 150-250nM) of a probe and/or (ii) l-50ng/mL (particularly 5-15ng/mL) of (yeast) tRNA. Other master mixes or master mix reagents, including other commercial master mixes which provides comparable assay performance may be substituted for the qPCRBIO Probe Mix Lo-Rox reagents.

O.lpl or more, such as 0.1-50 pl, particularly l-10pls of the amplification product from the first round may be added to the second round master mix. A preferred second round amplification master mix may comprise or consist of the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems) with 600 nM of the second (reverse) LTR primer, 600 nM of a tag primer, 200 nM of a probe and 10 ng/ml yeast tRNA. 2pl of the amplification product from the first round may be added to the second round master mix.

A LAMP master mix may comprise any suitable reagent(s), examples of which are readily (commercially) available, such as the PCR Biosystems mastermixes detailed earlier or LAMP-specific variants thereof, the WarmStart® Colorimetrix LAMP 2x Master Mix by New England Biolabs (https://www.rieb.uk.com/products/neb-catalogue/dria-amplificatiori/warmstart-lt;sup-gt;- amp;reg;-lt; sup gt; colorimetric-lamp- x-master- mix (dna amp;amp; ma)) or the Superscript IV RT- LAMP Mastermix by ThermoFisher Scientific (https://www.thermofisher.com/uk/en/home/life- science/pcr/rt-lamp-master-mix-isothermal-amplification.html) X. Other master mixes or master mix reagents, including other commercial master mixes which provides comparable assay performance may also be used.

A LAMP master mix may comprise or consist of a FIP, a BIP, an F3 and/or a B3 primer (typically all of a FIP, BIP, an F3 and a B3 primer), and optionally an LF and/or LB, optionally further comprising or consisting of (i) a carrier DNA or RNA (e.g. tRNA, particularly yeast tRNA). By way of non-limiting example, a master mix may comprise 10 to lOOpM (e.g. 40pM) of each of the Fl and Bl primers, 2 to 50pM (e.g. lOpM) of each of the F3, B3, Loop F and Loop B primers (where loop primers are included) and 10 to lOOpM (e.g. 50uM) of a colorimetric (or stain) solution. The components may be reconstituted in molecular grade water.

Samples

A method or assay of the invention may be carried out on a sample obtained from a subject (including non-human species - including plants). In some preferred embodiments the subject is human. The sample may be any suitable biological material, such as a cellular material or fluid. By way of non-limiting example a sample may comprise or consist of blood, plasma, saliva, serum, sputum, hair, urine, cerebral spinal fluid (CSF), vaginal swab material, oral or nasal swab material, cells, a cellular extract, a tissue sample, a tissue biopsy, a stool sample and the like. The precise biological sample that is taken from the subject may vary, but the sampling preferably is minimally invasive and is easily performed by conventional techniques. The sample type may be selected depending on the virus to be detected and/or quantified. By way of non-limiting example, for viruses such as HIV-1, the sample may be a whole blood sample, spinal or other bodily fluid, a purified peripheral blood leukocyte sample or other cellular subtype, a cells type sorted from bulk leukocytes or other tissue, the subject's peripheral blood mononuclear cells (PBMCs) or a lysates thereof. The sample may be taken from the subject before, during, and/or after treatment for a disease. The sample may be an archival sample that has been frozen, paraffin embedded, dehydrated or preserved and/or stored by any other method. The sample may be taken from a subject suspected of being infected with a particular virus, for example if the subject is symptomatic for infection with said virus.

As described herein, the methods of the invention allow for the detection and/or quantification of integrated virus. As such, methods of the invention can be carried out on cellular material, e.g. cell lysates or particular cellular compartments. Any suitable cell type may be used, as the sample for use in methods and assays of the invention. The cell type may be selected on the basis of the virus to be detected and/or quantified. By way of non-limiting example, for HIV-1, a sample may comprise or consist of peripheral blood mononuclear cells (PBMCs) or PBMC lysate.

As described herein, the methods of the invention allow for the detection and/or quantification of total virus nucleic acid (i.e., all RNA and DNA species produces by the target species). Any suitable sample type may be used for such methods and assays of the invention. The sample may be selected on the basis of the virus to be detected and/or quantified. By way of non-limiting example, for HIV-1, a sample may comprise or consist of blood, plasma, serum or CSF.

SEQUENCE INFORMATION

SEQ ID NO: 1 - forward primer round 1 (semi-nested RNA or DNA)

ATGCCACGTAAGCGAAACTTCAATAAAGCTTGCCTTGA

SEQ ID NO: 2 - reverse primer (total RNA or DNA)

AGGGATCTCTAGITACCA

SEQ ID NO: 3 - forward primer (non-nested RNA or DNA)

TCAATAAAGCTTGCCTTGA

SEQ ID NO: 4 - forward primer round 2 (semi-nested RNA or DNA, tag/tag primer)

ATGCCACGTAAGCGAAACT SEQ ID NO: 5 - probe (total RNA or DNA)

ACAGAYGGGCACACACIACT

SEQ ID NO: 6 - HIV-1 LTR

CAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGG

GAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACT

CTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGC

SEQ ID NO: 7 - SCP1 random probe sequence

CTGGGTAGAGTAGTCACAGAATGCG

SEQ ID NO: 8 - HPRT-1 cDNA

TGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAAAGATGGTCAAGGTCGCA

AGCTTGCTGGTGAAAAGGACC

SEQ ID NO: 9 - HPRT forward primer

TGACACTGGCAAAACAATGCA

SEQ ID NO: 10 - HPRT reverse primer

AGCTTGCTGGTGAAAAGGACC

SEQ ID NO: 11 - HPRT VIC/TAMRA probe

TTTCCTTGGTCAGGCAGTATAATC

SEQ ID NO: 12 - HXB2 LTR 496-516

GGCTAACTAGGGAACCCACTG

SEQ ID NO: 13 - HXB2 LTR 522-546

CACTCAAGGCAAGCTTTATTGAGGC

SEQ ID NO: 14 - HXB2 LTR 572-549

CACAACAGACGGGCACACACTTGA SEQ ID NO: 15 - HXB2 LTR 599-582

TGGTAICTAGAGATCCCT

SEQ ID NO: 16 - HXB2 LTR 599-622

TCCACACTGACTAAAAGGGTCTGA

SEQ ID NO: 17 - HXB2 GAG 652-633

CAGTCGCGCCCGAACAGGGA

SEQ ID NO: 18 - HXB2 LTR 633-609

GCTAGAGATTTTCCACACTGACTAA

SEQ ID NO: 19 - HXB2 GAG 652-680

ACCTGAAAGCGAAAGGGAAACCAGAGGAG

SEQ ID NO: 20 - HXB2 GAG 680-699

GAGTCCTGCGTCGAGAGAGC

SEQ ID NO: 21 - HXB2 LTR sequence (from bp 1 to 633)

TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTT

CCCTGATTAGCAGAACTACACACCAGGGCCAGGGATCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTA

GTACCAGTTGAGCCAGAGAAGTTAGAAGAAGCCAACAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGC

CTGCATGGAATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCAC

ATGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGACATCGAGCTTGCTACAAGGGACTTTCCGCTG

GGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCA

GCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGG

AACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTC

TGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGC

SEQ ID NO: 22 - HXB2 4529-5096 (integrase gene)

TCTTTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAATACATACTGACAATGGCAGCAATTTCACCGGTGCT

ACGGTTAGGGCCGCCTGTTGGTGGGCGGGAATCAAGCAGGAATTTGGAATTCCCTACAATCCCCAAAGTCAA

GGAGTAGTAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTT

AAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCA GGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATT

CAAAATTTTCGGGTTTATTACAGGGACAGCAGAAATCCACTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAG

GTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATT

AGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAG

SEQ ID NO: 23 - HXB2 2445-5096 (conserved region of polymerase gene)

GAAATCTGTGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATC

TGTTGACTCAGATTGGTTGCACTTTAAATTTTCCCATTAGCCCTATTGAGACTGTACCAGTAAAATTAAAGCCA

GGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGT

ACAGAGATGGAAAAGGAAGGGAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCC

ATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGAC

TTCTGGGAAGTTCAATTAGGAATACCACATCCCGCAGGGTTAAAAAAGAAAAAATCAGTAACAGTACTGGAT

GTGGGTGATGCATATTTTTCAGTTCCCTTAGATGAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTAT

AAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAAT

ATTCCAAAGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTTATCTATCAATACA

TGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAGCTGAGACAAC

ATCTGTTGAGGTGGGGACTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTA

TGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAAGACAGCTGGACTGTCAATGAC

ATACAGAAGTTAGTGGGGAAATTGAATTGGGCAAGTCAGATTTACCCAGGGATTAAAGTAAGGCAATTATGT

AAACTCCTTAGAGGAACCAAAGCACTAACAGAAGTAATACCACTAACAGAAGAAGCAGAGCTAGAACTGGCA

GAAAACAGAGAGATTCTAAAAGAACCAGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAA

ATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGA

AAATATGCAAGAATGAGGGGTGCCCACACTAATGATGTAAAACAATTAACAGAGGCAGTGCAAAAAATAACC

ACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAACTGCCCATACAAAAGGAAACATGGGAAACA

TGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTTAATACCCCTCCCTTAGTGAAAT

TATGGTACCAGTTAGAGAAAGAACCCATAGTAGGAGCAGAAACCTTCTATGTAGATGGGGCAGCTAACAGGG

AGACTAAATTAGGAAAAGCAGGATATGTTACTAATAGAGGAAGACAAAAAGTTGTCACCCTAACTGACACAA

CAAATCAGAAGACTGAGTTACAAGCAATTTATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTAAC

AGACTCACAATATGCATTAGGAATCATTCAAGCACAACCAGATCAAAGTGAATCAGAGTTAGTCAATCAAATA

ATAGAGCAGTTAATAAAAAAGGAAAAGGTCTATCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAA

TGAACAAGTAGATAAATTAGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCA

AGATGAACATGAGAAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTGCCACCTGTAGTAGCA

AAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGAGAAGCCATGCATGGACAAGTAGACTGTAGT

CCAGGAATATGGCAACTAGATTGTACACATTTAGAAGGAAAAGTTATCCTGGTAGCAGTTCATGTAGCCAGTG GATATATAGAAGCAGAAGTTATTCCAGCAGAAACAGGGCAGGAAACAGCATATTTTCTTTTAAAATTAGCAGG

AAGATGGCCAGTAAAAACAATACATACTGACAATGGCAGCAATTTCACCGGTGCTACGGTTAGGGCCGCCTG

TTGGTGGGCGGGAATCAAGCAGGAATTTGGAATTCCCTACAATCCCCAAAGTCAAGGAGTAGTAGAATCTAT

GAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAAT

GGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAG

ACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTAT

TACAGGGACAGCAGAAATCCACTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGT

AATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATTAGGGATTATGGAAAACA

GATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAG

SEQ ID NO: 24 - HXB2 sequence

TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTT

CCCTGATTAGCAGAACTACACACCAGGGCCAGGGATCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTA

GTACCAGTTGAGCCAGAGAAGTTAGAAGAAGCCAACAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGC

CTGCATGGAATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCAC

ATGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGACATCGAGCTTGCTACAAGGGACTTTCCGCTG

GGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCA

GCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGG

AACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTC

TGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACC

TGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGA

GGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGT

GCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAA

AGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCC

TGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAG

AACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAA

GGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACA

CAGGACACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCAAATGGTACATCAGG

CCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGATACC

CATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACAT

CAAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGAATGGGATAGAGTGCATCCAGTG

CATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTAC

CCTTCAGGAACAAATAGGATGGATGACAAATAATCCACCTATCCCAGTAGGAGAAATTTATAAAAGATGGATA

ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGACCAAAG GAACCCTTTAGAGACTATGTAGACCGGTTCTATAAAACTCTAAGAGCCGAGCAAGCTTCACAGGAGGTAAAAA

ATTGGATGACAGAAACCTTGTTGGTCCAAAATGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGACC

AGCGGCTACACTAGAAGAAATGATGACAGCATGTCAGGGAGTAGGAGGACCCGGCCATAAGGCAAGAGTTT

TGGCTGAAGCAATGAGCCAAGTAACAAATTCAGCTACCATAATGATGCAGAGAGGCAATTTTAGGAACCAAA

GAAAGATTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACACAGCCAGAAATTGCAGGGCCCCTAGGAAAA

AGGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATGAAAGATTGTACTGAGAGACAGGCTAA I I I I I I AG

GGAAGATCTGGCCTTCCTACAAGGGAAGGCCAGGGAATTTTCTTCAGAGCAGACCAGAGCCAACAGCCCCAC

CAGAAGAGAGCTTCAGGTCTGGGGTAGAGACAACAACTCCCCCTCAGAAGCAGGAGCCGATAGACAAGGAA

CTGTATCCTTTAACTTCCCTCAGGTCACTCTTTGGCAACGACCCCTCGTCACAATAAAGATAGGGGGGCAACTA

AAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAGAAGAAATGAGTTTGCCAGGAAGATGGAAA

CCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGATCAGATACTCATAGAAATCTGT

GGACATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTC

AGATTGGTTGCACTTTAAATTTTCCCATTAGCCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGAT

GGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAGATG

GAAAAGGAAGGGAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAA

AAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGACTTCTGGGAA

GTTCAATTAGGAATACCACATCCCGCAGGGTTAAAAAAGAAAAAATCAGTAACAGTACTGGATGTGGGTGAT

GCATATTTTTCAGTTCCCTTAGATGAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGA

GACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAAAG

TAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTTATCTATCAATACATGGATGATT

TGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAGCTGAGACAACATCTGTTGA

GGTGGGGACTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCA

TCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAAGACAGCTGGACTGTCAATGACATACAGAA

GTTAGTGGGGAAATTGAATTGGGCAAGTCAGATTTACCCAGGGATTAAAGTAAGGCAATTATGTAAACTCCTT

AGAGGAACCAAAGCACTAACAGAAGTAATACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAG

AGAGATTCTAAAAGAACCAGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAA

GCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAATATGC

AAGAATGAGGGGTGCCCACACTAATGATGTAAAACAATTAACAGAGGCAGTGCAAAAAATAACCACAGAAA

GCATAGTAATATGGGGAAAGACTCCTAAATTTAAACTGCCCATACAAAAGGAAACATGGGAAACATGGTGGA

CAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTTAATACCCCTCCCTTAGTGAAATTATGGTAC

CAGTTAGAGAAAGAACCCATAGTAGGAGCAGAAACCTTCTATGTAGATGGGGCAGCTAACAGGGAGACTAA

ATTAGGAAAAGCAGGATATGTTACTAATAGAGGAAGACAAAAAGTTGTCACCCTAACTGACACAACAAATCA

GAAGACTGAGTTACAAGCAATTTATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTAACAGACTCA

CAATATGCATTAGGAATCATTCAAGCACAACCAGATCAAAGTGAATCAGAGTTAGTCAATCAAATAATAGAGC AGTTAATAAAAAAGGAAAAGGTCTATCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAA

GTAGATAAATTAGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGATGAA

CATGAGAAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTGCCACCTGTAGTAGCAAAAGAAA

TAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGAGAAGCCATGCATGGACAAGTAGACTGTAGTCCAGGAA

TATGGCAACTAGATTGTACACATTTAGAAGGAAAAGTTATCCTGGTAGCAGTTCATGTAGCCAGTGGATATAT

AGAAGCAGAAGTTATTCCAGCAGAAACAGGGCAGGAAACAGCATATTTTCTTTTAAAATTAGCAGGAAGATG

GCCAGTAAAAACAATACATACTGACAATGGCAGCAATTTCACCGGTGCTACGGTTAGGGCCGCCTGTTGGTG

GGCGGGAATCAAGCAGGAATTTGGAATTCCCTACAATCCCCAAAGTCAAGGAGTAGTAGAATCTATGAATAA

AGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGT

ATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAA

TAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAG

GGACAGCAGAAATCCACTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATAC

AAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATTAGGGATTATGGAAAACAGATG

GCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAGAACATGGAAAAGTTTAGTAAAACACCATAT

GTATGTTTCAGGGAAAGCTAGGGGATGGTTTTATAGACATCACTATGAAAGCCCTCATCCAAGAATAAGTTCA

GAAGTACACATCCCACTAGGGGATGCTAGATTGGTAATAACAACATATTGGGGTCTGCATACAGGAGAAAGA

GACTGGCATTTGGGTCAGGGAGTCTCCATAGAATGGAGGAAAAAGAGATATAGCACACAAGTAGACCCTGAA

CTAGCAGACCAACTAATTCATCTGTATTACTTTGACTGTTTTTCAGACTCTGCTATAAGAAAGGCCTTATTAGGA

CACATAGTTAGCCCTAGGTGTGAATATCAAGCAGGACATAACAAGGTAGGATCTCTACAATACTTGGCACTAG

CAGCATTAATAACACCAAAAAAGATAAAGCCACCTTTGCCTAGTGTTACGAAACTGACAGAGGATAGATGGA

ACAAGCCCCAGAAGACCAAGGGCCACAGAGGGAGCCACACAATGAATGGACACTAGAGCTTTTAGAGGAGC

TTAAGAATGAAGCTGTTAGACATTTTCCTAGGATTTGGCTCCATGGCTTAGGGCAACATATCTATGAAACTTAT

GGGGATACTTGGGCAGGAGTGGAAGCCATAATAAGAATTCTGCAACAACTGCTGTTTATCCATTTTCAGAATT

GGGTGTCGACATAGCAGAATAGGCGTTACTCGACAGAGGAGAGCAAGAAATGGAGCCAGTAGATCCTAGAC

TAGAGCCCTGGAAGCATCCAGGAAGTCAGCCTAAAACTGCTTGTACCAATTGCTATTGTAAAAAGTGTTGCTT

TCATTGCCAAGTTTGTTTCATAACAAAAGCCTTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGA

AGAGCTCATCAGAACAGTCAGACTCATCAAGCTTCTCTATCAAAGCAGTAAGTAGTACATGTAACGCAACCTA

TACCAATAGTAGCAATAGTAGCATTAGTAGTAGCAATAATAATAGCAATAGTTGTGTGGTCCATAGTAATCAT

AGAATATAGGAAAATATTAAGACAAAGAAAAATAGACAGGTTAATTGATAGACTAATAGAAAGAGCAGAAG

ACAGTGGCAATGAGAGTGAAGGAGAAATATCAGCACTTGTGGAGATGGGGGTGGAGATGGGGCACCATGCT

CCTTGGGATGTTGATGATCTGTAGTGCTACAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCTGTGTGG

AAGGAAGCAACCACCACTCTATTTTGTGCATCAGATGCTAAAGCATATGATACAGAGGTACATAATGTTTGGG

CCACACATGCCTGTGTACCCACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACAGAAAATTTTAA

CATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGATATAATCAGTTTATGGGATCAAAGCCTAAAGCC ATGTGTAAAATTAACCCCACTCTGTGTTAGTTTAAAGTGCACTGATTTGAAGAATGATACTAATACCAATAGTA

GTAGCGGGAGAATGATAATGGAGAAAGGAGAGATAAAAAACTGCTCTTTCAATATCAGCACAAGCATAAGA

GGTAAGGTGCAGAAAGAATATGCATTTTTTTATAAACTTGATATAATACCAATAGATAATGATACTACCAGCTA

TAAGTTGACAAGTTGTAACACCTCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCAATTCCCATAC

ATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAATGTAATAATAAGACGTTCAATGGAACAGGACCATGTAC

AAATGTCAGCACAGTACAATGTACACATGGAATTAGGCCAGTAGTATCAACTCAACTGCTGTTAAATGGCAGT

CTAGCAGAAGAAGAGGTAGTAATTAGATCTGTCAATTTCACGGACAATGCTAAAACCATAATAGTACAGCTGA

ACACATCTGTAGAAATTAATTGTACAAGACCCAACAACAATACAAGAAAAAGAATCCGTATCCAGAGAGGACC

AGGGAGAGCATTTGTTACAATAGGAAAAATAGGAAATATGAGACAAGCACATTGTAACATTAGTAGAGCAAA

ATGGAATAACACTTTAAAACAGATAGCTAGCAAATTAAGAGAACAATTTGGAAATAATAAAACAATAATCTTT

AAGCAATCCTCAGGAGGGGACCCAGAAATTGTAACGCACAGTTTTAATTGTGGAGGGGAATTTTTCTACTGTA

ATTCAACACAACTGTTTAATAGTACTTGGTTTAATAGTACTTGGAGTACTGAAGGGTCAAATAACACTGAAGG

AAGTGACACAATCACCCTCCCATGCAGAATAAAACAAATTATAAACATGTGGCAGAAAGTAGGAAAAGCAAT

GTATGCCCCTCCCATCAGTGGACAAATTAGATGTTCATCAAATATTACAGGGCTGCTATTAACAAGAGATGGT

GGTAATAGCAACAATGAGTCCGAGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGA

ATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGT

GCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTA

TGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACA

ATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGC

AAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACT

CATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACG

ACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAA

ACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACA

TAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTT

TGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCC

CGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCG

ATTAGTGAACGGATCCTTGGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTG

AGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATAT

TGGTGGAATCTCCTACAGTATTGGAGTCAGGAACTAAAGAATAGTGCTGTTAGCTTGCTCAATGCCACAGCCA

TAGCAGTAGCTGAGGGGACAGATAGGGTTATAGAAGTAGTACAAGGAGCTTGTAGAGCTATTCGCCACATAC

CTAGAAGAATAAGACAGGGCTTGGAAAGGATTTTGCTATAAGATGGGTGGCAAGTGGTCAAAAAGTAGTGT

GATTGGATGGCCTACTGTAAGGGAAAGAATGAGACGAGCTGAGCCAGCAGCAGATAGGGTGGGAGCAGCAT

CTCGAGACCTGGAAAAACATGGAGCAATCACAAGTAGCAATACAGCAGCTACCAATGCTGCTTGTGCCTGGCT

AGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACA AGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAA GACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGG GCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGATAGA AGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGA GAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGT ACTTCAAGAACTGCTGACATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCT GGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCT CTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGC TTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTT TTAGTCAGTGTGGAAAATCTCTAGCA

SEQ ID NO: 25 - Alul primer sequence

CC CAG CTA CTG GGG AGG CTG AGG

SEQ ID NO: 26 - Alu2 primer sequence

GCC TCC CAA AGT GCT GGG ATT ACA G

EXAMPLES

The invention is now described with reference to the Examples below. These are not limiting on the scope of the invention, and a person skilled in the art would be appreciate that suitable equivalents could be used within the scope of the present invention. Thus, the Examples may be considered component parts of the invention, and the individual aspects described therein may be considered as disclosed independently, or in any combination.

Materials and Methods

The Polymerase Chain Reaction or PCR forms the basis for HIV-1 viral measurement in blood or other fluids and within cellular compartments of the human body. The exemplified method targets specific regions of HIV-1 nucleic acids and amplifies them exponentially with detection via a fluorescent read-out.

Assay Design

Oligonucleotide sequence names were denoted by the first nucleotide of their HXB2 reference sequence base position and an "F" for forward primer, "P" for probe or "R" for reverse primer. The main oligonucleotide sets studied during this project were also denoted by the first author and their year of publication e.g., Brussel 2005. Probes were initially prepared in a TaqMan PCR format with doubly quenched Iowa-Zen chemistry (www.idtdna.com).

Previously Reported Laboratory Developed Assays (LDAs)

Initial selection of assay oligonucleotides was based on assessment of candidate oligonucleotides for broad cross-subtype specificity against all available sequences in the United States Los Alamos National Medical Library's (LANL) HIV-1 database (www.hiv.lanl.gov) using the QuickAlign tool.

Modified Laboratory Developed Assays

To improve cross-subtype specificity, a modified oligonucleotide set - 525F/574P/599R, was designed by using AllelelD software v7.0 (PREMIER Biosoft, San Francisco, CA, USA) (http://www.premierbiosoft.com/bacterial-identification/index.html) against an alignment of the highly homologous region running from 433 to 633 base pairs of the HIV-1 long terminal repeat (LTR) from the LANL HIV-1 compendium database

(https://www.hiv.lanl.gov/content/sequence/HIV/COMPENDIUM/compendium.html). The oligonucleotide selection was finalised by using the Integrated DNA Technology online oligoanalyzer tool (https://eu.idtdna.com/pages/tools/oligoanalyzer) to select the sequences that provided the best in silico assay parameters from the possible permutations.

Analysis of the finalized sequences against the compendium database showed very few mismatches (data not shown). These mismatches were accounted for by replacements with a wobble base and inosine modified bases at relevant positions.

To allow maximum utility HIV-1 assays were developed in both a standard and a semi-nested format, using both DNA and RNA targets. The semi-nested format allows target enrichment of limited samples and thus increases assay sensitivity. The RTqPCR format for quantification of HIV-1 RNA in fluids, including plasma viral load, was optimised, and validated using synthetic plasmid templates and WHO-recognized international standards. The qPCR format, for quantification of total nucleic acids (i.e. RNA and DNA) in cellular compartments, was optimized and validated using synthetic plasmid templates and crude lysates from 8E5 cells, a recognized cell-line standard.

Synthetic Target Plasmid Templates for Assay Development

HIV-1 LTR DNA Template

The following sequence from 433 to 633bp of the HIV-1 LTR was used: - CAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGG GAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACT CTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGC (SEQ ID NO: 6)

The fragment was cloned into a pMA-T vector at the Sfil cloning site by the Life Technologies GeneArt Service (ThermoFisher Scientific). Plasmid DNA was purified from transformed E. coll K12 (dam+dcm+tonA) (Figure 2a), linearised using a single-cutter Scal-HF® Restriction Enzyme (New England BioLabs, Ipswich, MA, USA) and purified using a PureLink™ Quick Gel Extraction Kit (Invitrogen/ThermoFisher Scientific, Waltham, MA, USA).

Extraction Control DNA Template

A DNA plasmid incorporating a previously described SCP1 random probe sequence - CTGGGTAGAGTAGTCACAGAATGCG flanked by the 496F (SEQ ID NO: 12) and 622R (SEQ ID NO: 16) HIV-1 primers was synthesised, via the ThermoFisher GeneArt service, using a manufacturer-selected cloning vector.

HIV-1 LTR and Extraction Control RNA Templates

RNA transcripts of the linearized DNA templates were synthesized using the MEGAscrip T7 Transcription Kit (Ambion/ThermoFisher Scientific). The size and purity of the products was confirmed by gel electrophoresis. The gel-purified transcripts were quantified using a NanoDrop™ 1000 spectrophotometer (ThermoFisher Scientific).

HPRT-1 Reference Gene cDNA Template

A template encoding the HPRT region targeted by an in-house RT-qPCR reference gene assay TGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAAAGATGGTCAAGGTCGCA AGCTTGCTGGTGAAAAGGACC was synthesized via the GeneArt service, using a manufacturer-selected cloning vector. RNA sample data was considered valid if the reference gene could be detected regardless of whether a positive HIV-1 signal was detected.

Synthetic Template Copy Number Calculation and Standard Generation

The copy number/pl for each synthetic template stock solution was determined from its molecular weight and Avogadro's constant using an online copy number calculator (http://www.endmemo.com/bio/dnacopynum.php). The stocks were diluted to working solutions of either lxl0⁸ or 3xl0⁸ copies/pl in Tris/EDTA pH 8.0 (TE) buffer for DNA or DEPC-treated water for RNA. The working solutions were divided into 5pl single-use aliquots in PCR tube strips and stored at -80°C. Single aliquots were broken off the strips and thawed before each use. Ten-fold standard curve dilutions ranging from 3xl0⁷ to 3 viral input copies were generally run in triplicate. More replicates were run at the lowest dilutions when determining the assay lower limit of detection (LLOD), lower limit of quantification (LLOQ), precision, and reproducibility.

Ethics

For the Kenyan donors from lAVI's protocol L ethical approval for sample collection and processing was granted by the Kenyatta National Hospital /University of Nairobi ethics and research committee (KNH/UON-ERC); study reference P69/03/2011.

For the Rwandese donors from lAVI's protocol L ethical approval for sample collection and processing was granted by the Rwanda National Ethics Committee (RNEC) Research Ethics Committee; study reference NO.1023/RNEC/2020

For the donor samples from the London St. Stephen's Trust ethical approval for sample collection and processing was granted by the NHS London-Chelsea Research Ethics Committee; study reference 96.ND14, RREC1108. Informed written consent was provided by all participants prior to obtaining each sample, and clinical data was collected as part of standard care.

The samples from the EQ.APOL and AIDS Reagent Program repositories have all been anonymized with no links back to the individual samples. They are exempt from regulations concerning human samples.

Sample Preparation

Crude Cellular DNA Lysates

For PBMC lysate preparation, cryovials containing frozen cells were thawed in a water bath at 37°C for approximately 2 minutes until only a small ice crystal remained. The cells were then transferred into 50ml pre-warmed RPMI containing 10% fetal calf serum, lOmM HEPES, 2mM L- glutamine, ImM sodium pyruvate and lx penicillin-streptomycin. Cells were pelleted at 250g for 10 minutes, supernatants decanted, and cells resuspended in 1ml PBS. Cell concentrations were determined using a Vi-cell counter (Beckman Coulter Instruments, Inc.). Cells were then made up to 1x10^s cells per ml in PBS, divided into 1ml aliquots in sample tubes and pelleted at 2500g for 5 minutes. Supernatants were aspirated twice by pipetting to ensure a very dry pellet before cell lysis. For 8E5 cell lysate preparation, frozen pre-made pellets containing lxlO⁷ cells were used (see section titled "Cellular Standards for DNA qPCR"). A lysis buffer was prepared using lOmM Tris HCL pH8 (Ambion), 50mM KCI (Ambion) and 0.4mg/ml Proteinase K (Qiagen) in molecular biology grade water. Forty-two pl of lysis buffer was added to the cell pellets and vortexed for 10 to 15 seconds. Cells were lysed for 3 hours at 55°C. To reduce DNA viscosity and break-up clumps, the following cycling protocol was then used: 65°C for 1 min, 96°C for 2 min, 65°C for 4 min, 96°C for 1 min, 65°C for 1 min, 96°C for 30 sec. Finally, lysates were incubated at 95°C for 15 minutes to ensure complete inactivation of the proteinase K. DNA was quantified using a NanoDrop 1000 spectrophotometer (ThermoFisher Scientific) and genome copy number/pl determined by molecular weight and Avogadro's constant using an online copy number calculator (http://www.endmemo.com/bio/dnacopynum.php).

To improve sensitivity and precision at the lowest target concentrations, DNA lysates were pre-digested with an EcoRI-HF enzyme (New England BioLabs, Ipswich, MA, USA), according to the manufacturer's instructions, before amplification (Figure lb). The EcoRI-HF enzyme, did not cut DNA within the PCR target region.

Plasma RNA Extraction

The QIAamp UltraSens Virus Kit (Qiagen) was used for viral RNA extraction from plasma samples with the following modifications: 1 ml of each HIV-1 spiked EDTA plasma sample was used; 3x10^s input copies of the extraction control template was added to the spin columns along with the lysed and proteinase-digested samples; the lysate and AW1 buffer were centrifuged at 8000g; all subsequent centrifugation steps were at 20,000g; only one final RNA elution step was performed using 30pl AVE buffer following a 5-minute incubation at room temperature.

Cellular Standards for DNA qPCR

8E5 cells are an established lymphocyte cell line that contain a single copy of integrated HIV- 1 DNA genome per cell. The 8E5 cells were obtained from the AIDS reagent program (https://www.hivreagentprogram.org Cat No. ARP 95) and passaged 3 times in RPMI containing 10% fetal calf serum, lOmM HEPES, 2mM L-glutamine, ImM sodium pyruvate and lx penicillinstreptomycin at 37°C, 5% CO₂. Cells were then pelleted at 250g for 10 minutes, washed in PBS and divided into 1ml aliquots of lxlO⁷ cells per cryovial. Cells were pelleted at 250g for 10 minutes then resuspended in 1ml of ice-cold Fetal Calf Serum containing 10% Dimethyl Sulfoxide (DMSO). The cryovials were frozen down to -80°C overnight in a rate-controlled Stratagene stratacooler and stored at -150°C in vapor phase liquid nitrogen.

To prepare cell pellets for the generation of PCR standard curves, an aliquot of each of the working stocks of the cells was thawed out as in the previous section when required and passaged 3 to 6 more times. The cell suspensions were then centrifuged at 250g for 10 minutes, quantified using a ViCell Counter (Beckman Coulter Instruments, Inc.) and adjusted to lxl0⁷ cells/ml in PBS. One ml aliquots of the cell suspensions were divided into cryovials, and the cells were pelleted at maximum speed for 10 minutes in a benchtop microfuge. Supernatants were carefully removed twice by pipetting, and the dried cell pellets stored at -80°C until required.

Instrument and Probe-Chemistry Optimization

A series of real-time PCR instruments including the Applied Biosystems 7500 Dx thermocycler, Applied Biosystems 7300 thermocycler, Applied Biosystems StepOnePlus thermocycler, Roche Lightcycler and finally the QuantiStudio 3 thermocycler (ThermoFisher Scientific) were evaluated with the assay. In all cases default manufacturer settings were used. The QuantiStudio 3 instrument and its manufacturer recommended Minor Grove Binding (MGB) TaqMan probe labeled with 6- CaboxyFlourescein at the 5' end and a non-fluorescent quencher (NFQ) at the 3' (6FAM/MGBNFQ) provided superior performance.

PCR Reagents and Protocols

For initial primer evaluation, the QuantiTect Probe kit (Qiagen) for qPCR of DNA targets and the Superscript III Platinum One-Step qRT-PCR Kit (ThermoFisher Scientific) for RNA targets, were used.

To shorten assay time using fast-cycling, the use of the following test kits and master mixes for qPCR was evaluated: - the QuantiFast Probe (Qiagen); Kapa Probe Fast (Merck); or qPCRBIO Probe Mix Lo-Rox (PCR Biosystems); the EXPRESS One-Step Superscript (ThermoFisher Scientific); and the qPCRBIO 1-Step Go Lo-Rox kit (PCR Biosystems), based on recommendations from PCR instrument manufacturers and colleagues.

To further increase assay sensitivity and reduce variability at low target concentrations, various concentrations of tRNA were added to the master-mix and optimized. A "step-up" cycling protocol was employed to improve sensitivity and cross-subtype specificity. "Step-up" cycling rescues PCR reactions where there are minor mismatches between the assay oligonucleotides and the target template species and serves to increase the sensitivity and precision for detection of HIV subtypes with minor sequence polymorphisms at low target concentrations.

Real-Time Quantitative PCR (qPCR) Protocol for DNA Targets

The final conditions selected for PCR amplification were the qPCRBIO Probe Mix Lo-Rox reagents (PCR Biosystems) with 600 nM Forward Primer, 600 nM Reverse Primer, 200 nM Probe; up to 3x10^s input-copies of enzyme-digested target HIV DNA; 10 ng/ml yeast tRNA (Sigma-Aldrich, MO, USA) added to the PCR master-mix, and a "step-up" cycling protocol with an initial activation step of 95°C for 15 min, 3 pre-amplification cycles of 94°C for 20 s, 52°C (8°C below the ideal annealing temperature) for 10 s and 60°C for 1 min, followed by 40 amplification cycles of 94°C for 20 s, 56°C (4°C below the ideal annealing temperature) for 10 s, 60°C for 1 min. Fluorescent data was collected at the 60°C amplification step of each of the 40 cycles (Figure 2b). Cell copy number in DNA samples was calculated relative to an RNaseP TaqMan copy number reference assay (Applied Biosystems).

Reverse Transcriptase Quantitative PCR (RT-qPCR) Protocol for RNA Targets

Standard Non-nested RTqPCR

A standard, non-nested, RT-qPCR protocol was used to determine the LLOQ. plasma viral load of the assay. The qPCRBIO 1-Step Go Lo-Rox kit (PCR Biosystems) was used with the same oligonucleotide and tRNA concentrations as in the qPCR protocol. An initial reverse transcription step of 50°C for 10 min was followed by a "touch up" cycling protocol with an enzyme activation step of 95°C for 2 min; 40 amplification cycles of 95°C for 10 s, 52°C -> 60°C* for 10 s and finally 72°C for 1 min. The arrow and asterisk indicates that the annealing temperature was increased from 52°C to 60°C in equal increments across 40 cycles. Five pl of sample was used in a final reaction volume of 50pl for maximum sensitivity.

Semi-nested RTqPCR for sample enrichment

To allow target enrichment of limited archival samples, a semi-nested RT-qPCR protocol was also developed (Figure 2c). A bacteriophage lambda sequence - a foreign, non-human oligonucleotide - was used as a tag on the first-round forward primer, X-525F, while a primer against the tag - XT, was used in the second round of the PCR. For this semi-nested protocol, HIV-1 cDNA synthesis and first round amplification were conducted using an Applied Biosystems Veriti Thermal Cycler (Applied Biosystems/ThermoFisher Scientific) and the qPCRBIO 1-Step Go Lo-Rox kit (PCR Biosystems) with 600 nM X_525F and 599R primers. A "step-up" cycling protocol was adopted with a reverse transcription step of 50°C for 15 min followed by a polymerase activation step of 95°C for 2 min; five amplification cycles of 95°C for 20 s; 52°C for 10 s and 60°C for 1 min; followed by seven cycles of 95°C for 20 s, 56°C for 10 s, and 60°C for 1 min. For second round amplification, 2 pL of the first-round product was used in the 20pL second round amplification (10%v/v), with 600 nM XT forward primer, 600 nM 599R reverse primer and 200 nM 574P probe. The same reagents, instrumentation and cycling protocol described for the qPCR of DNA targets were used. The presence of RNA in sample extracts was confirmed using an in-house HPRT1 reference gene assay and the HPRT synthetic template described previously to generate the standard curves. The qPCRBIO 1-Step Go Lo-Rox Kit reagents (PCR Biosystems) and the following cycling parameters were used with 900 nM TGACACTGGCAAAACAATGCA HPRT Forward Primer, 900 nM AGCTTGCTGGTGAAAAGGACC HPRT Reverse Primer and 250 nM TTTCCTTGGTCAGGCAGTATAATC VIC/TAMRA Probe (ThermoFisher Scientific). qPCR and RTqPCR Run Acceptance Criteria

Each assay run included quantification standards, and a known positive sample or an AcroMetrix HIV-1 positive control or calibrator (ThermoFisher Scientific); and a known negative sample or an AcroMetrix HIV-1 negative control (ThermoFisher Scientific). Assay runs were considered acceptable if no target was detected in the non-template and negative controls, the standard curve had a slope of -3.2 to -3.5, and R² values were >0.95. Linearity was based on the slope and R² of the standard curve and expressed as y = mx. The linear range of the assay was determined from the standard curves. Precision/reproducibility was expressed as a percentage of the coefficient of variability (CV), with a CV of less than 10% being acceptable. HIV-1 RNA target runs were only considered valid if a parallel HPRT1 reference gene assay was also valid and HPRT1 was detected in the samples, while HIV-1 DNA target runs were only considered valid if a parallel RNaseP reference gene assay was also valid and RNaseP was detected in the samples.

Assessing Laboratory Developed Assay Performance

Confirmation of Correct Sequence Amplification

To determine whether a single PCR product of the desired length and sequence was being produced by the assays, PCR products were run on a 1% Agarose gel (Fluka, NJ, USA). Bands were visualized using the SYBR Safe DNA Gel Stain (Invitrogen) and a Bio-Rad Molecular Imager Gel Doc XR+ with Imager Lab Software (Bio-Rad Laboratories) and cut out on a safe Imager 2.0 (ThermoFisher Scientific). DNA was recovered using a QIAquick gel purification kit (Qiagen). Purified PCR products were sequenced by the Sanger sequencing service at Genewiz, Surrey, United Kingdom and confirmed proper amplification of the target sequence.

Assay Linearity, Lower Limit of Detection (LLOD) and Lower Limit of Quantification with 95% confidence (LLOQ95) Linearity of the HIV-1 LDA was evaluated in a non-nested format on triplicate samples of the AcroMetrix HIV-1 Quantification Panel (ThermoFisher Scientific) ranging from 5xl0² to 5x10^s copies/ml.

LLOD/LLOQ95 values were calculated based on detection of two-fold dilutions of negative EDTA plasma samples spiked with the 5xl0⁴ copies/ml Acrometri standard (ThermoFisher Scientific) at dilutions ranging from 25 to 0.46 copies per ml with 10 replicates used per data point.

Intra-assay and Inter-assay Reproducibility and Precision

Cell pellet aliquots were thawed and lysed and quantified as previously described. Ten-fold serial dilutions ranging from 3x10^s to 3 input cell equivalents per reaction were prepared in TE Buffer containing lOOpg/ml salmon sperm DNA (ThermoFisher Scientific).

Intra-assay reproducibility was evaluated on 3 separate batches of serially diluted 8E5 cell lysates tested by a single technician on 3 separate occasions, using the standard qPCR protocol. Interassay reproducibility was evaluated on a single batch of 8E5 cells run on three separate occasions by three separate technicians. Mean values, within-run %CV, run-to-run %CV and total %CV were derived for each concentration in the dilution series. Six replicates per run were tested at the 3 input cells dilution, with 3 replicates per run at the other dilutions.

The precision of the assay was evaluated using the replicates from the intra- and inter-assay variability experiments. The means, standard deviations and coefficients of variation were calculated for the 30 replicates run at 3 copies, and 15 replicates run at higher dilutions.

Assay Specificity for HIV-1

The specificity of the assay for HIV-1 was determined using the standard non-nested RT-qPCR RNA protocol by demonstrating lack of a specific signal in the EDTA plasma from 10 uninfected donors (Biological Specialty Corporation, Colmar, PA) and lack of cross-reactivity with donor or spiked samples infected with other RNA viruses, including Affymetrix-Valiquant HBV and HCV standards (Affymetrix Inc., Santa Clara, CA), cultured EBV, HSV-1, CMV, VZV and Parvovirus from Zeptometrix (Buffalo, NY) spiked into Basematrix diluent (SeraCare, Milford, MA, USA), HIV-2 NIH Reference samples, and a College of American Pathologists (CAP) respiratory panel, ID2-A 2012, containing Influenza, Parainfluenza, RSV, Adenovirus, Human Metapneumovirus and Coronavirus also spiked into Basematrix diluent (SeraCare, Milford, MA).

The specificity of the assay on crude cellular lysates was determined using 74 HIV-1 negative IAVI protocol L donors (26 male and 46 female), 12 chronically infected protocol L donors (8 male and 4 female) and 32 treatment-suppressed male HIV-1 positive donors from the London St. Stephens Trust. The IAVI protocol L study was designed for assay characterization and testing of sample collection methods. Volunteers were enrolled from Kigali, Rwanda and the Kenyatta National Hospital and Kangemi Health Centre in Kenya where subtype A is predominant, followed by D, C and G. The London St. Stephens Trust participants were all confirmed as subtype B (supplementary table 5). Assay sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were determined using the online calculator - https://www.medcalc.org/calc/diagnostic_test.php.

HIV-1 Cross-Subtype Specificity

The cross-subtype specificity and accuracy of the assay was determined using well- characterized diversity panels from the U.S. Military's HIV Research Program (MHRP) and EQAPOL. The MHRP isolates represented an established 60-member international panel consisting of 10 isolates from each of the 6 Major HIV-1 subtypes (https://www.hivreagentprogram.org, Catalog Number ARP 11412). The EQAPOL isolates represented a broad range of recently sourced transmitter/founder (T/F) viral strains of HIV-1 including all major subtypes, common circulating recombinant forms (CRFs) and a few unique recombinant forms (URFs) (EQAPOL, duke.edu). The custom panel included all the transmitted/founder (T/F) viral strains that had been provided to EQAPOL by IAVI and that could be cultured to high titers in primary PBMCs. In total, 127 isolates were tested and comprised of 18 Subtype A, 22 Subtype A recombinants, 18 Subtype B, 17 Subtype C, 2 Subtype C recombinants, 13 Circulating Recombinant F01_AE (CRF01_AE), 12 Subtype D, 2 Subtype Fl, 2 Subtype F2, 12 Subtype G, 3 Subtype O and 6 Unique Recombinant Forms (URFs). The EQAPOL- supplied stock samples were diluted 1000-fold in AcroMetrix EDTA plasma dilution matrix (ThermoFisher Scientific) to bring them into the dynamic range of the assay. HIV-1 viral load was expressed in copies per ml, using concentrations determined by the AcroMetrix HIV-1 quantification reference panel (ThermoFisher Scientific).

Statistical Analysis

All data was analyzed using GraphPad Prism 9 (https://www.graphpad.com/scientific- software/pris) and Microsoft Excel unless otherwise stated. A variety of online calculators and tools were utilized wherever they are stated. PCR standard curve statistics were generated by the instrument manufacturer's software packages.

Example 1: Primer and Probe Design

Oligonucleotide sequence names are denoted by the first nucleotide of their HXB2 reference sequence base position and an "F" for forward primer, "P" for probe or "R" for reverse primer. Known oligonucleotide sets studied during this project are denoted by the first author and their year of publication e.g., Brussel 2005 (Human Retrovirus Protocols METHODS IN MOLECULAR BIOLOGY™ 304 (2005).

A new set of oligonucleotides was designed using AllelelD 7.0 AllelelD software v7.0 (PREMIER Biosoft, San Francisco, CA, USA) (http://www.premierbiosoft.com/bacterial- identification/index.html) against an alignment of the highly homologous region running from 433 to 633 base pairs of the HIV-1 long terminal repeat (LTR) from the LANL HIV-1 compendium database (https://www.hiv.lanl.gov/content/sequence/HIV/COMPENDIUM/compendium.html). The oligonucleotide selection was finalized by using the Integrated DNA Technology online oligoanalyzer tool (https://eu.idtdna.com/pages/tools/oligoanalyzer) to select the sequences that provided the best in silico assay parameters from the possible permutations.

Analysis of the finalized sequences against the compendium database showed very few mismatches (Table 1). These mismatches were accounted for by replacements with a wobble base and inosine modified bases at relevant positions (Table 2). Probes were initially prepared in a TaqMan PCR format with doubly quenched Iowa-Zen chemistry (www.idtdna.com)

Table 1: Summary of results from the alignment of the primer probe set used in the current assay as compared to those of the Brussel 2005 (Freidrich 2010 internal Reverse Primer), Vandergeeten 2014, van der Sluis 2013, Schvachsa 2007 or Viard 2004 oligonucleotides against the HIV compendium database.

N = number of sequences examined including at least one (up to 6) isolates from each of subtype A, B, C, D, CRFO1_AE, F, CRF02_AG, and G. The % of isolates that have one or no mismatches or more than 1 mismatch in the indicated primer is shown.

Brussel 2005 = Human Retrovirus Protocols METHODS IN MOLECULAR BIOLOGY™ 304; Friedrich 2010 = Virol. J. 7:354; Vandergeeten 2014 = J. Virol. 88(21): 12385-12396; van der Sluis 2013 = Virol. Met. 187: 94-102;

Schvachsa 2007 = J. Virol. Met. 140: 222-227; Viard, 2004 = Medecine et Maladies Infectieuses 34, S209-S215

Table 2: Oligonucleotides designed and tested for various formats of the HIV-1 assay a Genbank Accession ID K03455. Inosine and mixed bases shown in enlarged bold font. AllelelD 7.0 and IDT Oligoanalyzer software utilized.

Assay optimization of the revised oligonucleotides was performed on a variety of instruments and finalized on a QuantiStudio 3™ thermocycler (ThermoFisher Scientific) using a major groove binding (MGB) probe containing a non-fluorescent quencher. The MGB group at the 3' end of the probe increased its melting temperature (Tm) while the NFQ component of the probe quenched the signal from the 6FAM fluorescent dye before hydrolysis, in a manner that resulted in an even lower background signal than a non-NFQ probe. The finalized protocol as described in the methods resulted in a greatly improved assay with a linear dynamic range of over 7 logs (Figures 2 and 3).

Example 2: Semi-Nested Quantification of HIV-1 LTRs is Improved Compared with Existing Nested Assay

Initial screening of published nucleotides found that the 496F/546P/633R primer-probe set (Brussel 2005), with an optional Friedrich 2010 internal reverse primer 622R produced the best and most consistent results but showed limited specificity for non-sub type B isolates on a sub-panel of 20 EQ.APOL isolates that were difficult to detect and accurately quantify (Table 3). Table 3 shows data on the 8 most problematic strains (in other words 8 HIV-1 strains that were could not be quantified or were difficult to quantify using the Brussel/Freidrich assay - 496F/546P/622R1/633R2, compared to Table 3: Effect of Nested PCR, Step-up cycling, and Oligonucleotide Sequence on Cross-Subtype Specificity and Accuracy of HIV-1 RT-qPCR LDAs.

Samples that were under-quantified by the LDAs; Bold type = Samples that were correctly quantified by the assay

the performance of the 525F/574P/599R primers/probes of the invention). lOng/ml tRNA was used in the master-mix, in all these experiments.

Further modifications were made to the assay protocol, including adjustment of oligonucleotide concentration, utilisation of nested PCR, step-up cycling, and addition of tRNA to the master-mix. This unique combination of additional modifications resulted in significantly improved assay performance with a standard curve of > 90% efficiency, a linear range over 5 logs and a LLOD/LLOQ95 of 3 input copies of HIV DNA (Table 3).

Subsequent sequence analysis of these oligonucleotide set against the HIV-1 LANL compendium database, showed significant mismatch issues (Supplementary Table 2) and suggesting that the unique oligonucleotides designed in Example 1 are required to achieve the observed improvements in assay performance.

Example 3: Instrument and Probe-Chemistry Refinement

Refinement of the revised oligonucleotides was performed on a variety of instruments and finalized on a QuantiStudio 3™ thermocycler (ThermoFisher Scientific) using a major groove binding (MGB) probe containing a non-fluorescent quencher. The MGB group at the 3' end of the probe increased its melting temperature (T_m) while the NFQ component of the probe quenched the signal from the 6FAM fluorescent dye before hydrolysis, in a manner that resulted in an even lower background signal than a non-NFQ probe. Accordingly, the finalised protocol as described in the above methods and using the unique primer/probes identified in Table 2 resulted in a significantly improved assay with a linear dynamic range of over 7 logs (Figures 2 and 3) over known LDAs and known oligonucleotides (e.g. Brussel/Freidrich). Gel electrophoresis of the qPCR products from the revised LDA produced bands of the expected size of 74 bp (599 - 525 = 74bp). Sequencing of the bands confirmed the amplification of the expected region.

The following further modifications further improved assay performance:

PCR Reagents and Protocols

The described step-up cycling protocol using 10 ng/ml tRNA provided greater sensitivity, linearity and precision when compared to universal cycling conditions (Figure 3a, Table 3).

Real-Time Quantitative PCR (qPCR) Protocol for DNA Targets

Decreasing reaction volumes to 20pl and maximum ramp rates or fast cycling was used, the PCR Biosystems Universal Probe (FAST) kit performed better than the QuantiFast™ Probe (Qiagen) and Kapa Probe Fast (Merck), providing a significant time and cost saving from the previously used Qiagen QuantiTect Probe kit.

Reverse Transcriptase Quantitative PCR (RT-qPCR) Protocol for RNA Targets

Standard non-nested RTqPCR

The standard non-nested format of the RTqPCR or viral load assay was adapted to the 1-Step Go mastermix (PCR Biosystems). It performed better than the EXPRESS One-Step Superscript kit (ThermoFisher Scientific) and as well as the Superscript II l/platinum kit when reaction volumes were decreased to 20pl and maximum ramp rates or fast cycling was used. This provided significant time and cost savings.

For samples with target concentrations below 2 copies per pl, using a 50pl final reaction volume and "touch up" cycling, increased assay sensitivity 10-fold with the 1-Step Go mastermix (PCR Biosystems).

Semi-nested RTqPCR for sample enrichment

The use of a semi-nested assay format allowed for target enrichment of limited samples and further improved assay sensitivity.

Example 4: Assay Linearity, Reproducibility, Precision, Lower Limit of Detection (LLOD) and Lower Limit of Quantification with 95% confidence (LLOQ95)

A linear relationship was obtained for the qPCR format of the revised LDA against 8E5 cell DNA over a range of lOxlO¹ to 10xl0⁷ input copies of HIV-1. This format of the assay was capable of reproducibly detecting the equivalent of a single infected cell (Figure 3b and c, Table 4).

Table 4: Precision of Assay as Measured Using on Dilutions of Crude Lysates of 8E5 Cells

Evaluation of the performance of the RT-qPCR format of the revised LDA on a synthetic plasmid RNA template showed good linearity, accuracy, and precision over a range of 100 to 5x10^s copies of HIV-1 RNA/ml (R² of 0.91) when evaluated on the AcroMetrix HIV-1 linearity panel (ThermoFisher Scientific) (Figure 3b, Figure 3c). The lower limit of detection (LLOD) was determined by probit regression of serial dilutions of EDTA plasma spiked with a HIV-1 AcroMetrix quantification standard near the cut-off level of the assay. The LLOD was found to be 88 copies/ml at the 95% level and 17 copies/ml at 50% (Figure 3d).

The precision of the assay was evaluated on serial dilutions of lysates of 8E5 cells testing over 15 replicates at 300,000 to 30 cell equivalents, and 30 replicates at 3 cell equivalents. Excellent reproducibility was observed at all dilutions, with the inter-assay coefficient of variation ranging from 1.9 - 5.8% and a CV of 3.1% at the 3 input copy level. Intra-assay variability which compares results obtained on different days was 3.7 +/- 2.5% (Table 4).

Example 5: The Assay Shows High Specificity for HIV-1

The assay was highly specific as demonstrated by a lack of signal in EDTA plasma from 10 uninfected donors (Biological Specialty Corporation) and a lack of reactivity with plasma from individuals infected with Influenza, Parainfluenza, RSV, Adenovirus, Human Metapneumovirus, Coronavirus, HBV, HCV, EBV, HSV-1, CMV, VZV, Parvovirus, HIV-2A and HIV-2B (Table 5).

Table 5: Specificity of Assay on Plasma Samples from Individuals Infected with Other Viruses or from

Uninfected Individuals.

The assay was determined to be 95.76% accurate with 100% sensitivity and 93.24% specificity on 44 HIV-1 positive and 74 HIV-1 negative crude cellular lysates. The samples for this component of the validation were derived from 74 HIV-1 negative donors from I AVI's Protocol L cohort (formerly the International AIDS Vaccine Initiative). They comprised of 26 male and 46 female participants. The HIV- 1 positive group contained 12 chronically infected protocol L donors (8 male and 4 female) and 32 cART-suppressed male HIV-1 positive donors from the London St. Stephens Trust. The protocol L donors come from regions of Rwanda and Kenya where HIV-1 subtype A is predominant, followed by D, C and G while the London participants were all confirmed as subtype B. The positive predictive value of the assay was 89.80% while the negative predictive value was 100% with this sample set (Table 6).

Example 6: HIV-1 Cross-Subtype Specificity

The LDA of the invention accurately detected and quantified 19 out of the 20 problem strains in the preliminary diversity panel (95%), missing only the CRF01_AE strain KC596065 (Table 3). The ability of the assay to provide accurate quantitative measurement of plasma viral load over a wide range of HIV-1 subtypes was then evaluated on a panel of 127 isolates from the External Quality Assurance Program Oversite Laboratory (EQAPOL) and the US Military's HIV Research Program (USMHRP) spiked into plasma samples. The assay detected all but one of the additional virus strains in the panel (Figure 3f) - a 2004 subtype C isolate from China (AY713414). Thus, the assay of the invention shows excellent cross-subtype specificity, with a 99.2% detection rate. A Bland-Altman plot showing the difference between the measurements obtained by the LDA of the invention compared with viral load values obtained by the commercial Roche Cobas

Table 6: Specificity of Assay on crude PBMC lysates from HIV-1 infected or uninfected individuals.

Calculations computed using https://www.medcalc.org/calc/diagnostic test.php.

Values in parentheses are the 95% confidence intervals

AmpliPrep/Cobas TaqMan HIV-1 test v2.0™ assay shows a tight relationship with an R2 of 0.03, representing parallel performance (Figure 3d), with a slight bias (0.388) for the assay of the invention (Figure 2e & g). The relationship between the LDA of the invention and the Roche assay by individual isolates as sorted by subtype is shown in Figure 4.

Discussion

The inventors have developed a highly sensitive, specific, and robust quantitative PCR assay for viral detection targeting viral LTR, as exemplified herein using unique primers and probes for the HIV- 1 LTR. These assays have potentially advantageous applications for in research and for quantifying viral levels in infected individuals, such as HIV-1 infected individuals.

The present inventors have developed different assay formats and protocols, including a traditional RT-qPCR format for viral load determination and a cell-based DNA format that can be used to monitor residual levels of viral nucleic acids in treatment-suppressed individuals. For HIV-1, the assay design included selection of primers, probes, and amplification conditions to allow sensitive detection of all major HIV-1 subtypes worldwide, including group M subtypes A (East Africa), C (Southern Africa, India, Nepal), D (Eastern and Central Africa), CRF01_A/E (Thailand), and CRF02_A/G (West Africa and Central Europe) and Group O.

A variety of thermocyclers and master mixes were evaluated in the process of development of this assay. Whilst the methods are exemplified using a Thermofisher Quantistudio 3.0 thermocycle, it is within the routine skill of one of ordinary skill in the art to make any modifications needed to use alternative thermocyclers to carry out the methods of the invention.

Other notable improvements made by the inventors include the adaptation of the MGB group at the 3' end of the probe allowed for lowering the melting temperature (T_m) and use of an NFQ component in the probe to quench the signal from the 6FAM fluorescent dye before hydrolysis, in a manner that results in an even lower background signal than a non-NFQ. probe.

Despite initially selecting the most promising oligonucleotide sequences that had been reported in the literature as being broadly cross-subtype specific, assessment of the Brussel 2005 assay with a Friedrich 2010 internal reverse primer, which performed exceptionally well on a synthetic template, found that it accurately quantified only 7 out of 20 (35%) strains of HIV-1 viruses tested in an initial cross-subtype EQ.APOL diversity sub-panel (Table 3). Interestingly, alignment of these oligonucleotide sequences against the published sequences of the panel members did not reveal any mismatches in the strains that were not detected by the assay. Alignment of these oligonucleotides against the compendium database, however, revealed several mismatch issues, particularly in subtype AE and O ( Table 1). This was unexpected and indicates that in-silico predictions of cross-subtype specificity based simply on sequence alignments can be misleading.

Before designing the revised oligonucleotide set, a rigorous alignment analysis of the Brussel/Friedrich oligonucleotide sequences was conducted against the current edition of the LANL compendium database (data not shown). The compendium database is comprised of 37 highly curated sequences from major HIV-1 group M subtypes (four sequences per subtypes); 32 CRFs and other reference sequences and 11 subtype N, O or CPZ. The database is designed to represent the diversity of the sequences present within the entire HIV-1 database but allows quicker and more accurate analysis of variation as repetition and poor sequence information is eliminated. The alignments against the compendium database more accurately reflected assay performance and identified multiple mismatch issues, particularly in subtype AE and O (Table 1). The LDA of the invention was therefore designed against the compendium database and then verified against the entire database. It corrected the mismatch issues found in the conventional Brussel/Friedrich oligonucleotides and incorporated a wobble and Inosine, a modified base, in two locations. As already stated, the resulting assay performed significantly better than the conventional Brussel/Friedrich assay on a large diverse panel of HIVj-1 strains.

To determine whether the discrepancy between general sequence alignment versus the compendium alignment was a common phenomenon, the inventors re-analysed other oligonucleotide sets reported in the literature as having good HIV-1 cross-subtype specificity against the compendium database; using the same method disclosed herein for design of the new LDA. The inventors surprisingly found that multiple assays reported in the art as highly sensitive and broadly cross reactive, actually exhibit less favourable base-pair identity statistics when analysed using the unique methodology of the invention.

The LTR region of HIV-1 is particularly problematic for accurate assay design because sequencing enzymes become error-prone and drop off at the end of genomes. Indeed, despite all the sequences being classified as "complete", there are many sequences with only partial or no information within the LTR region, even in the carefully curated compendium database. The inventors therefore propose a new approach for the design of viral detection assays using LTR targets, particularly for HIV-1. Specifically, the inventors have devised a screening and design method for oligonucleotide primer and/or probes which comprises analysis of compendium databases, rather than the entirety of reported sequences, and that this results in good cross-subtype specificity.

The variability of the viral genomes, particularly the HIV-1 genome makes it very challenging to find three well-conserved regions of 25-35 nucleotides (two for the primers and one for the internal probe) within 200 base pairs of each other, for use with TaqMan PCR. The rationale behind the step- up or touch-up amplification methods was to allow for less stringent annealing at lower initial temperatures, followed gradually by higher more stringent annealing temperatures. The addition of yeast tRNA to PCR master-mixes and sample diluents significantly improved assay efficiency, quantification sensitivity and precision at low viral levels, such as are found in treated subjects.

The semi-nested format of the assay, with a tag on the forward primer in the first round and a primer against this tag in the second round, allowed for the use of only three regions and increased the chance of good cross-subtype coverage. This format allows for target enrichment and is recommended for limited samples. Rigorous cleanliness and great care must be taken to avoid crosscontamination with the semi-nested assay format. The use of a "tag" on one of the primers coupled with less stringent initial annealing temperatures, allowed for amplifications across regions of incomplete homology which increased the chance of good cross-subtype coverage. The tag subsequently allowed amplification of only specific amplicons from the first round by reducing nonspecific amplification of any primer-dimers or nonspecific artefacts generated in the primary PCR thus maintaining PCR specificity.

The use of crude DNA lysis makes the qPCR format of the assay easy and affordable. It maximizes assay sensitivity by preventing the unnecessary loss of precious sample through more complicated DNA extraction procedures.

Detection of very low levels of residual viral sequences as may be present in cART-treated individuals with undetectable viral loads has important applications for predicting re-emergence of infectivity or evaluation of approaches to viral cure.

The LDA of the invention could be combined with an assay against conserved regions of the integrase or polymerase gene to further improve its cross-subtype specificity.

The LDA of the invention is highly sensitive with excellent cross-subtype specificity and has the potential to play an important role in HIV-1 research and in improving clinical outcomes if used correctly. Using semi-nested qPCR, for target enrichment, allows for highly sensitive detection, when compared to non-nested qPCR and increases the likelihood of detecting ultra-low levels of HIV-1 within samples. This assay provides a convenient, sensitive, specific, and reproducible measure of HIV-1 viral RNA in plasma and HIV-1 viral RNA and DNA in PBMC of patients under anti-HIV therapy. The assay is suitable for monitoring the efficacy of therapeutic strategies and for measurement of viral persistence in support of studies aimed at testing the efficacy of vaccines, antiretroviral combinations, and HIV-1 eradication strategies.

Claims

1. An in vitro method of detecting and/or quantifying a virus, which method comprises the steps of: a) identifying an oligonucleotide region that is conserved across multiple different strains of a virus by aligning a diversity panel of a plurality of different strains of said virus; b) designing one or more primer sequence that is suitable for amplification of the identified nucleotide region, wherein said one or more primer sequence is specific to the identified nucleotide region, wherein any mismatches between the sequences of the plurality of different strains identified by the alignment are corrected; and c) detecting and/or quantifying the virus within a biological sample by amplifying viral nucleic acid using the one or more primer sequence; wherein optionally the diversity panel comprises sequences from at least 20 strains of said virus.

2. An in vitro method of detecting and/or quantifying a virus, which method comprises the steps of: a) amplifying viral nucleic acid in a biological sample with PCR using a first and second primer for a viral LTR sequence, wherein the first and second primers hybridise to different regions within the LTR sequence, and wherein said first primer is linked to a tag sequence; b) subjecting the amplified nucleic acid from step (a) to another amplification with PCR using the second primer and a primer for the tag sequence; c) detecting and/or quantifying the nucleic acid that was amplified through steps (a) and (b), wherein the detected/quantified nucleic acid correlates with the number of copies of the virus genome.

3. The method of claim 2, wherein the first and/or second primer comprises at least one inosine base.

4. The method of claim 2 or , wherein the first and second primers hybridise to different regions of the 5' LTR sequence.

5. The method of claim 4, wherein: a) the first primer hybridises to the R region of the 5' LTR sequence; and/or b) the second primer hybridises to the U5 region of the 5' LTR sequence.

6. The method of claim 4 or 5, wherein: a) the first primer is SEQ ID NO: 1, or a variant differing by one or two nucleotides from SEQ ID NO: 1; and/or b) the second primer is SEQ ID NO: 2, or a variant differing by one or two nucleotides from SEQ ID NO: 2.

7. The method of any one of the preceding claims, which is used to detect and/or quantify total viral RNA and/or DNA.

8. An in vitro method of detecting and/or quantifying an integrated virus, which method comprises the steps of: a) amplifying viral nucleic acid in a biological sample with PCR using a primer for an Alu sequence and a first primer for a viral LTR sequence, wherein said primer for a viral LTR sequence is linked to a tag sequence; b) subjecting the amplified nucleic acid from step (a) to another amplification with PCR using a primer for the tag sequence and a second primer for the viral LTR sequence; c) detecting and/or quantifying the nucleic acid that was amplified through steps (a) and (b), wherein the detected/quantified nucleic acid correlates with the number of copies of the integrated virus genome.

9. The method of claim 8, wherein the Alu sequence primer, the first primer for the viral LTR sequence and/or the second primer for the viral LTR sequence comprises at least one inosine base.

10. The method of claim 8 or 9, wherein: a) the first primer for the viral LTR sequence hybridises to the R region of the 5' LTR sequence; and/or b) the second primer for the viral LTR sequence hybridises to the U5 region of the 5' LTR sequence.

11. The method of claim 10, wherein: a) the first primer for the viral LTR sequence may comprise or consist of a nucleic acid selected from SEQ ID NO: 1 (or a variant differing by one or two nucleic acids from SEQ ID NO: 1) and SEQ ID NO 3 (or a variant differing by one or two nucleotides from SEQ ID NO: 3); and/or b) the second primer for the viral LTR sequence may comprise or consist of a nucleic acid of SEQ ID NO: 2, or a variant differing by one or two nucleotides from SEQ ID NO: 2.

12. The method of any one of the preceding claims, wherein the virus is a retrovirus or lentivirus, wherein optionally the retrovirus is HIV, preferably HIV-1.

13. The method of any one of the preceding claims, wherein the tag sequence or the primer for the tag sequence: a) is a sequence not found in the virus or host organism; and/or b) comprises or consists of a bacteriophage lambda nucleotide sequence, optionally SEQ ID NO: 4, or a variant differing by one or two nucleotides from SEQ ID NO: 4.

14. The method according to any one of claims 2 to 13, wherein the PCR of step (b) is performed in the presence of at least one detectable probe that specifically hybridises with the viral nucleic acid amplified, wherein the hybridisation of the probe allows for the detection and/or quantification of the virus.

15. The method of claim 14, wherein the probe comprises: a) comprises a fluorescent moiety; b) comprises a 5' FAM, VIC, TET or NED dye, and optionally a 3' non-fluorescent quencher (NFQ), wherein preferably said probe further comprises a 3' minor groove binder moiety (MGB); and/or c) comprises or consists of SEQ ID NO: 5, or a variant differing by one or two nucleotides from SEQ ID NO: 5, and preferably wherein said probe is 5'6-

FAM/ACAGAYGGGCACACACIACT/MGBNFQ-3'. The method of any one of the preceding claims, wherein the sample comprises fluid and/or cells from a subject, preferably wherein said sample comprises: a) plasma; b) peripheral blood mononuclear cells (PBMCs); and/or c) PBMC lysate. The method of any one of claims 2 to 16, wherein: a) step (a) comprises from 10 to 15 cycles of PCR; and/or b) step (b) comprises from 30 to 50 cycles of PCR. The method of any one of claims 2 to 17, wherein: a) the PCR cycles are step-up PCR cycles; b) the PCR is qPCR or RTqPCR; c) a ten-fold dilution is carried out between steps (a) and (b); d) each PCR reaction comprises tRNA, optionally at a concentration of 10 ng/mL; and/or e) the lower limit of detection of the target organism is (i) about 80 to 90 copies/mL, or (ii) about 3 input copies of nucleic acid. The method of any one of claims 2 to 18, wherein one or more primer sequence is designed using the method of identifying and designing a primer sequence as defined in steps (a) and (b) of claim 1. The method of any one of the preceding claims which is a multiplex method in which one or more additional target region of the viral genome is detected and/or quantified, wherein optionally said one or more additional target region is selected from a viral integrase gene and/or a viral polymerase gene. An oligonucleotide primer which comprises or consists of any one of SEQ ID NOs: 1 to 4 or a variant differing by one or two nucleotides from any one of SEQ. ID NOs: 1 to 4. An oligonucleotide probe which comprises or consists of SEQ ID NO: 5, or a variant differing by one or two nucleotides from SEQ. ID NO: 5. A set of oligonucleotides comprising: a) (i) SEQ ID NO 3, or a variant differing by one or two nucleotides from SEQ ID NO: 3; or (ii) SEQ ID NO: 1, or a variant differing by one or two nucleotides from SEQ ID NO: 1 and SEQ ID NO: 4, or a variant differing by one or two nucleotides from SEQ ID NO: 4; b) SEQ ID NO 2, or a variant differing by one or two nucleotides from SEQ ID NO: 2; and c) SEQ ID NO: 5, preferably 5'6-FAM/ACAGAYGGGCACACACIACT/MGBNFQ-3', or a variant differing by one or two nucleotides from SEQ ID NO: 5. A kit for performing a method of any one of claims 2 to 20, which comprises: a) (i) a first container containing a first and second primer for a viral LTR sequence and a second container containing the second primer for a viral LTR sequence and a primer for the tag sequence; or

(ii) a first container containing a first primer for a viral LTR sequence, a second container containing a second primer for a viral LTR sequence, and a third container containing a primer for the tag sequence; and

(iii) a detectable probe, wherein the detectable probe may be contained in same container as the second primer and/or tag primer, or in a separate container; or b) (i) a first container containing an Alu primer and a first primer for a viral LTR sequence and a second container containing a second primer for a viral LTR sequence and a primer for the tag sequence; or

(ii) a first container containing an Alu primer, a second container containing a first primer for a viral LTR sequence, a third container containing a second primer for a viral LTR sequence, and a fourth container containing a primer for the tag sequence; and

(iii) a detectable probe, wherein the detectable probe may be contained in same container as the second primer for a viral LTR sequence and/or tag primer, or in a separate container; wherein optionally said kit may comprise one or more additional reagent for carrying out said method. An in vitro method of detecting and/or quantifying a virus, comprising carrying out PCR using one or more oligonucleotides primers as defined in claim 21 and/or one or more oligonucleotide probe as defined in claim 22, wherein preferably said method comprises carrying out PCR using a set of oligonucleotides as defined in claim 23, and wherein optionally said PCR is: a) qPCR or RT-qPCR; and/or b) nested or semi-nested PCR.