WO2008062385A2 - Antiretroviral drug resistance testing - Google Patents

Abstract

The invention relates to a test to detect mutations associated with antiretroviral drug resistance in HIV-1 using an allele-specific real-time PCR assay. The method uses mutation- specific PCR primers designed to detect and quantify the subset of HIV-1 viruses carrying the nucleic acid changes which are associated with cause resistance. The invention provides primers, primer sets, and amplification conditions which offer increased sensitivity of detection, after having designed and tested many different primers, primer sets, and PCR conditions. In addition, the method of the invention can be used to estimate the relative proportion of resistant viruses in the total virus population in a host. This method is therefore suited for the detection and quantification of enfuvirtide, protease, and reverse transcriptase resistance mutations in HIV-1 and will provide more useful clinical information for patient management.

Description

ANTIRETROVIRAL DRUG RESISTANCE TESTING

FIELD OF THE INVENTION

This invention relates to antiretroviral drug resistance testing. More particularly, this invention relates to methods of determining and quantifying drug resistance in a virus population

(Human Immunodeficiency Virus Type-1 ), as well as primers and primer sets for use in determining such drug resistance.

BACKGROUND OF THE INVENTION

The introduction of antiretroviral therapy has played a major role in the treatment of HIV-1 infected people all over the world. However, many of the successes achieved have been negated by the emergence of antiretroviral drug resistance. Resistant viruses develop naturally during the replication of the virus and can usually only be prevented from arising by complete suppression of replication using antiretroviral drug therapy. Often, however, poor adherence to drug therapies or other factors leads to the natural selection of drug resistant viruses that can compromise the health of the patient and allow transmission of resistant viruses to other persons.

Currently there are two methods of which the inventors are aware for detecting drug resistance in virus populations. The first method (genotyping) involves identifying mutations in viral nucleic acid by performing nucleic acid sequencing, while the second method (phenotyping) involves direct measurement of the susceptibility of a virus population to antiretroviral drugs in vitro.

The most common viral enzymes known to the Applicants that are targeted by antiretroviral drugs are (i) reverse transcriptase, (ii) protease (iii) integrase; as well as the envelope structural proteins (iv) gp120 and (v) gp41. The two classes of drugs that interfere with the reverse transcriptase enzyme, are called nucleoside reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI). Different types of resistance mutations develop against these drugs (discrimination, excision and binding).

The protease generally shows resistance mutations in or around the active site, detrimentally affecting inhibitor binding.

In HIV-1 , drug resistance arises as a result of positive selection of minority species of viruses carrying drug resistance mutations in essential viral proteins such as reverse transcriptase and protease, both of which are encoded by the HIV-1 pol gene. These changes occur naturally as a result of copying errors introduced into the pol gene during the RNA to DNA conversion process carried out by reverse transcriptase (reverse transcription). Drug resistance is mediated by amino- acid changes in the molecular structure of reverse transcriptase and/or protease enzymes. These changes are a result of nucleotide changes in the genetic code that specifies which amino-acid should be incorporated into the assembly of viral proteins.

To detect such mutations, conventional methods known to the inventors rely on nucleic acid sequencing of the HIV-1 pol gene that codes for reverse transcriptase and protease. A limitation of this method is the low level of sensitivity of detection. The altered DNA product is not detectable at a frequency of less than 10-20% of the total population. Also, the method is not quantitative and does not estimate the proportion of viruses carrying the mutations.

An additional new class of antiretroviral drugs known to the inventors are called entry inhibitors. These drugs target the entry or fusion process of HIV-1. A current fusion inhibitor called

Enfuvirtide (also known as FUZEON, or T-20) specifically inhibits virus entry into a host cell by preventing the fusion process of the virus envelope and the host cell membrane, thereby preventing access of the virus core (containing viral RNA) into the cell cytoplasm. Enfuvirtide functions by binding to the first heptad repeat region (HR1 ) of HIV-1 gp41 , preventing the fusion process that involves binding of HR1 to the second heptad repeat region (HR2) from taking place. However,

HIV-1 can circumvent inhibition by Enfuvirtide by introducing mutations into the HR1 binding region and compensatory mutations in the HR2 and gp120 V3 (third variable) regions. It is thus an object of this invention to provide a method and detection means for identifying and quantifying specific antiretroviral drug resistance mutations in HIV-1 viral populations to facilitate clinical decisions regarding antiretroviral treatment options and protocols for management of HIV-1 positive people.

SUMMARY OF THE INVENTION

According to one aspect of the invention, there is provided an oligonucleotide primer having a sequence selected from the group including any one of SEQ ID NO 1 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention further provides a set of oligonucleotide primers, wherein the set includes at least one oligonucleotide primer having a sequence selected from any one of SEQ ID NO 1 to 159 inclusive; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention further provides a set of oligonucleotide primers, wherein the set includes at least one forward primer having a sequence selected from SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention further provides a set of oligonucleotide primers, wherein the set includes at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 1 19; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention further provides a set of oligonucleotide primers, wherein the set includes at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID NO 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention further provides a set of oligonucleotide primers, wherein the set includes at least one forward primer having a sequence of SEQ ID NO 152 to 155, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 156 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The, or each, oligonucleotide primer may contain modified bases, particularly Locked

Nucleic Acid (LNA) modifications at the 3' end thereof.

The invention also provides a method of detecting the presence of a virus, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence selected from any one or more of SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention further provides a method of detecting the presence of a virus, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention also provides a method of detecting the presence of a virus, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention also provides a method of detecting the presence of a virus, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 152 to 155, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 156 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention also provides a method of detecting a mutation in a virus population, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence selected from any one or more of SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention also provides a method of detecting a mutation in a virus population, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof. The invention also provides a method of detecting a mutation in a virus population, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention also provides a method of detecting a mutation in a virus population, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 152 to 155, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 156 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention also provides a method of detecting a plurality of mutations in a virus population, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence selected from any one or more of SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention also provides a method of detecting a plurality of mutations in a virus population, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention also provides a method of detecting a plurality of mutations in a virus population, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention also provides a method of detecting a plurality of mutations in a virus population, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 152 to 155, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 156 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention also provides a method for quantifying the levels of a virus population in a specimen, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence selected from any one or more of SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and subjecting the results of the polymerase chain reaction to ΔCT testing.

The invention also provides a method for quantifying the levels of a virus population in a specimen, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and subjecting the results of the polymerase chain reaction to ΔCT testing .

The invention also provides a method for quantifying the levels of a virus population in a specimen, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and subjecting the results of the polymerase chain reaction to ΔCT testing.

The invention also provides a method for quantifying the levels of a virus population in a specimen, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 152 to 155, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 156 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and subjecting the results of the polymerase chain reaction to ΔCT testing.

The invention also provides a method for quantifying the level of a mutation in a virus population relative to another mutation or a wild-type reference, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence selected from any one or more of SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention also provides a method for quantifying the level of a mutation in a virus population relative to another mutation or a wild-type reference, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention also provides a method for quantifying the level of a mutation in a virus population relative to another mutation or a wild-type reference, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention also provides a method for quantifying the level of a mutation in a virus population relative to another mutation or a wild-type reference, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 152 to 155, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 156 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

According to yet another aspect of the invention, there is provided a method of screening for mutations associated with abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, zidovudine, delavirdine, efavirenz and nevirapine resistance in a host, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence selected from any one or more of SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID

NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and screening the amplification product for the presence of mutations associated with resistance to any one or more of the antiretroviral drugs.

According to yet another aspect of the invention, there is provided a method of screening for mutations associated with resistance in a host to any one or more antiretroviral drugs selected from atazanavir, fosamprenavir, darunavir, indinavir, lopinavir, nelfinavir, saquinavir, tiprannavir and ritonavir, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and screening the amplification product for the presence of mutations associated with resistance to any one or more of the antiretroviral drugs.

According to yet another aspect of the invention, there is provided a method of screening for mutations associated with enfuvirtide resistance in a host, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and screening the amplification product for the presence of mutations associated with resistance to any one or more of the antiretroviral drugs.

The method may include the step of screening simultaneously for resistance to all of the abovementioned drugs.

According to yet another aspect of the invention, there is provided a method of quantifying levels of abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, zidovudine, delavirdine, efavirenz and nevirapine or atazanavir, fosamprenavir, darunavir, indinavir, lopinavir, nelfinavir, saquinavir, tiprannavir and ritonavir or enfuvirtide resistance in a host, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence selected from any one or more of SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and/or alternatively or additionally, at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID

NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and/or alternatively or additionally, at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of

SEQ ID NO 120 to 129 or SEQ ID 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and/or alternatively or additionally, at least one forward primer having a sequence of SEQ ID NO 152 to 155, and at least one reverse primer having a sequence selected from any one or more of

SEQ ID NO 156 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and quantifying for the presence of mutations associated with resistance in the amplification product to any one or more of the antiretroviral drugs using semi-quantitative PCR.

The method may include the step of screening simultaneously for resistance to all of the abovementioned drugs.

The invention further extends to a kit for the detection of mutations in a virus population, the kit including: at least one forward primer having a sequence selected from any one or more of SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and/or at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and/or at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and/or at least one forward primer having a sequence of SEQ ID NO 152 to 155, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 156 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The invention further extends to a kit for the relative quantification of mutations in a virus population, the kit including: at least one forward primer having a sequence selected from any one or more of SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and/or at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and/or at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and/or at least one forward primer having a sequence of SEQ ID NO 152 to 155, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 156 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and amplification means for the amplification of a nucleic product by polymerase chain reaction using the aforementioned primers.

The kit may include extraction means for the extraction of nucleic acid from specimens obtained from potentially diseased hosts.

The invention extends also to an artificial HIV-1 nucleic acid sequence, the nucleic acid sequence having homology to any one or both of SEQ ID NO 160 or SEQ ID NO 161 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

The virus or virus population may be of the genus Retroviridae. The virus may be selected from the group including HIV-1 , HIV-2 and SIV, and sub-types thereof. In one particular embodiment of the invention, the virus may be HIV-1 and various groups and sub-types thereof.

The mutations may be any one or more of the following mutations present in HIV populations, particularly HIV-1 populations:

^*Mutations shown above are indicated as wild-type amino acid/position/amino acid change.

The mutations may be linked to drug resistance in HIV populations, such as resistance to drugs which inhibit or prevent HIV infection or propagation by acting on the gp41 , reverse transcriptase, or protease proteins/enzymes.

The sequence identifier (SEQ ID 161 ) used in this specification is measured relative to a consensus pol DNA sequence generated by the inventors using published HIV-1 pol sequences available in the Genbank sequence database. The sequence identifier (SEQ ID 160) used in this specification is measured relative to a consensus env DNA sequence (gp41 ) generated by the inventors using published HIV-1 env sequences available in the Genbank sequence database.

The step of extracting nucleic acid from potentially diseased hosts may include the use of standard nucleic extraction techniques known to those skilled in the art.

The method of detecting or quantifying the levels of a virus population in a specimen may further include the step of quantifying the relative levels of amplification product using semi- quantitative PCR. The semi-quantitative PCR may be done in a real-time PCR instrument commercially available under the trade name ABI7900HT.

The virus populations may be obtained from specimens, such as whole blood specimens from infected hosts. The viral nucleic acid, in the form of RNA, may be extracted using an automated nucleic acid extraction instrument and an extraction kit.

Furthermore, the viral RNA may, prior to screening, be converted to DNA. The RNA to DNA conversion may be performed in an ABI 7900 HT real-time PCR instrument using the reverse transcriptase enzyme and the primer sets of the invention. Following conversion into complementary DNA, the converted nucleic acids may be amplified using the primer sets of the invention, together with a SYBR green master mix.

The method of the invention furthermore may include the step of utilizing the initial DNA product obtained hereinbefore for the detection of the absolute number of RNA particles using a standard curve method.

Advantageously, mutation detection in an HI virus population may be accomplished in an allele specific manner using the primers of the invention. Mutation detection may be performed in silico or in vitro, typically in a 96 or 384 well plate.

Wild-type and drug resistance mutations may be detected and discriminated using an ABI

7900 HT machine. The proportion of wild-type and mutant virus particles may be calculated using the ΔCT method. Advantageously, the proportion of wild-type to mutant viruses may additionally be related back to the viral load, to calculate the absolute number of mutant or wild-type viruses in a total population.

Further features of the invention will now become apparent from the following description, by way of example only, with reference to the accompanying drawings and figures.

DRAWINGS

In the drawings: Figure 1 shows a standard curve of protease primer set (pr1 F1 and prR1 ) of the invention used for viral load determination and to generate a fragment suitable for mutation analysis.

Figure 2 shows a standard curve of Reverse Transcriptase primer set (rt1 F2 and rtR7) of the invention used for viral load determination and to generate a fragment suitable for mutation analysis.

Figure 3 shows a standard curve of Reverse Transcriptase primer set (rt2F1 and rtR5) of the invention used for viral load determination and to generate a fragment suitable for mutation analysis.

Figure 4 shows a standard curve of a gp41 resistance primer set (Fin3/Rin) of the invention used for viral load determination and to generate a fragment suitable for mutation analysis.

SEQUENCE LISTING

Primer sequences

HIV-I REVERSE TRANSCRIPTASE PRIMERS

SEQ ID NO 1 CrtM41LlA 5' TGTAATTTTTCCTTCCTTCTCCAA

SEQ ID NO 2 CrtM41LlG 5' TGTAATTTTTCCTTCCTTCTCCAG

SEQ ID NO 3 CrtM41MlT 5' TGTAATTTTTCCTTCCTTCTCCAT

SEQ ID NO 4 CrtK65KlT 5 ' CTCCACTTAGTACTGTCCTTCTT

SEQ ID NO 5 CrtK65RlG 5 ' CTCCACTTAGTACTGTCCTTCTG

SEQ ID NO 6 CrtK65K2T 5 ' TCTCCACTTAGTACTGTCCTTCT

SEQ ID NO 7 CrtK65R2C 5 ' TCTCCACTTAGTACTGTCCTTCC

SEQ ID NO 8 CrtK70KlT 5 'ATCTACTAATTTTCTCCACT

SEQ ID NO 9 CrtK70RlC 5 'ATCTACTAATTTTCTCCACC

SEQ ID NO 10 CrtK70K2T 5 'ATCTACTAATTTTCTCCACTT

SEQ ID NO 11 CrtK70R2G 5 ΆTCTACTAATTTTCTCCACTG

SEQ ID NO 12 CrtK70E2C 5 'ATCTACTAATTTTCTCCACTC

SEQ ID NO 13 CrtK103NlG 5' CACATCCAGTACTGTCACTGATTTG

SEQ ID NO 14 CrtK103NlA 5' CACATCCAGTACTGTCACTGATTTA

SEQ ID NO 15 CrtK103KlT 5' CACATCCAGTACTGTCACTGATTTT

SEQ ID NO 16 CrtK103KlC 5' CACATCCAGTACTGTCACTGATTTC SEQ ID NO 17 CrtVlOβVIC 5 ' CACATCCAGTACTGTCAC

SEQ ID NO 18 CrtV106MlT 5 ' CACATCCAGTACTGTCAT

SEQ ID NO 19 CrtV106V2A 5 ' CCACATCCAGTACTGTCA

SEQ ID NO 20 CrtV106A2G 5 ' CCACATCCAGTACTGTCG

SEQ ID NO 21 CrtY181ClC 5' CCTACATACAAGTCATCCATATATTGAC

SEQ ID NO 22 CrtY181YlT 5' CCTACATACAAGTCATCCATATATTGAT

SEQ ID NO 23 CrtY188C2A 5 ΆTTTCTAAGTCAGATCCTACATA

SEQ ID NO 24 CrtY188Y2T 5 'ATTTCTAAGTCAGATCCTACATT

SEQ ID NO 25 CrtG190AlG 5' TGTTGCCCTATTTCTAAGTCAGATG

SEQ ID NO 26 CrtG190GlC 5' TGTTGCCCTATTTCTAAGTCAGATC

SEQ ID NO 27 CrtL210VlC 5' GGTGTGGTAAATCCCCACTTTAC

SEQ ID NO 28 CrtL210LlA 5' GGTGTGGTAAATCCCCACTTTAA

SEQ ID NO 29 CrtL210LlG 5' GGTGTGGTAAATCCCCACTTTAG

SEQ ID NO 30 CrtT215SlA 5' GATGTTTCTTGTCTGGTGTGGA

SEQ ID NO 31 CrtT215TlT 5' GATGTTTCTTGTCTGGTGTGGT

SEQ ID NO 32 CrtT215I2A 5' TGATGTTTCTTGTCTGGTGTGA

SEQ ID NO 33 CrtT215T2G 5' TGATGTTTCTTGTCTGGTGTGG

SEQ ID NO 34 CrtT215Y2T 5' TGATGTTTCTTGTCTGGTGTGT

SEQ ID NO 35 CrtK219QlG 5' GGGGTTCTTTCTGATGTTTCTG

SEQ ID NO 36 CrtK219KlT 5' GGGGTTCTTTCTGATGTTTCTT

SEQ ID NO 37 CrtM230MlT 5 'ATGGAGTTCATACCCCAT

SEQ ID NO 38 CrtM230LlA 5 'ATGGAGTTCATACCCCAA

SEQ ID NO 39 CrtM230LlG 5 'ATGGAGTTCATACCCCAG

SEQ ID NO 40 CrtlF2 (forward) 5 ' GGATGGCCCAAAGGTTAAACAATGG

SEQ ID NO 41 Crt2Fl (forward) 5 ' TGGATGTGGGGGATGCATATTTTTC

SEQ ID NO 42 CrtR5 (reverse) 5 'AGCTGTATAGGCTGTACTGTCC

SEQ ID NO 43 CrtR7 (reverse) 5 ΆTATTGCTGGTGATCCTTTCCATCC

SEQ ID NO 44 CrtD67NΔ 5 'AATTTTCTCCACTTAGTACTGTT

SEQ ID NO 45 CrtD67DΔ 5 'AATTTTCTCCACTTAGTACTGTC

SEQ ID NO 46 CrtT69AlCΔ 5 ' TAATTTTCTCCACTTAGC

SEQ ID NO 47 CrtT69SlAΔ 5 ' TAATTTTCTCCACTTAGA

SEQ ID NO 48 CrtT69TlTΔ 5 ' TAATTTTCTCCACTTAGT

SEQ ID NO 49 CrtT69S2CΔ 5 ' TCTACTAATTTTCTCCACTTAC

SEQ ID NO 50 CrtT69N2TΔ 5 ' TCTACTAATTTTCTCCACTTAT

SEQ ID NO 51 CrtT69T2GΔ 5 ' TCTACTAATTTTCTCCACTTAG

SEQ ID NO 52 CrtM184VlCΔ 5'ATCCTACATACAAGTCATCCAC

SEQ ID NO 53 CrtM184MlTΔ 5'ATCCTACATACAAGTCATCCAT

SEQ ID NO 54 CrtL210W2CΔ 5' TGTGGTAAATCCCCACTTTC SEQ ID NO 55 CrtL210L2AΔ 5' TGTGGTAAATCCCCACTTTA

SEQ ID NO 56 CrtY188ClC* 5 ' TTCTAAGTCAGATCCTACAC

SEQ ID NO 57 CrtY188YlT* 5 ' TTCTAAGTCAGATCCTACAT

SEQ ID NO 58 CrtY188LlA* 5 ' TTCTAAGTCAGATCCTACAA

SEQ ID NO 59 CrtlFl ( forward) 5 'ATTAAAGCCAGGAATGGATGGCCC

SEQ ID NO 60 CrtlF3 (forward) 5 'AAACTGTACCAGTAAAATTAAAGCCAGGAATG

SEQ ID NO 61 CrtlF4 (forward) 5 ΆCCAGTAAAATTAAAGCCAGGAATGGATGG

SEQ ID NO 62 Crt2F2 (forward) 5 ' TGAAGGCTTCAGGAAATATACTGCATTCAC

SEQ ID NO 63 Crt2F3 (forward) 5 ' TTCAGGAAATATACTGCATTCACCATACCTAG

SEQ ID NO 64 Crt3Fl (forward) 5 'AGAACCCCCATTTCTTTGGATGG

SEQ ID NO 65 Crt3F2 (forward) 5 ' TCAGAAAGAACCCCCATTTCTTTGG

SEQ ID NO 66 CrtRl (reverse) 5 ' CAGGATGGAGTTCATACCCCATCCA

SEQ ID NO 67 CrtR2 (reverse) 5 ' CCCTGGGTAAATCTGACTTGCCCA

SEQ ID NO 68 CrtR3 (reverse) 5 ' CCCTGTTCTCTGCCAATTCTAATTCTGC

SEQ ID NO 69 CrtR4 (reverse) 5 ΆTCTAAAGGAACTGAAAAATATGC

SEQ ID NO 70 CrtR6 (reverse) 5 ' CTTGGTAAATTTGATATGTCCATTG

SEQ ID NO 71 CrtR7x (reverse) 5 ' GGAATATTGCTGGTGATCCTTTCCATCC

SEQ ID NO 72 CrtR8 (reverse) 5 ' TTTCTGGCAGCTGTATAGGCTGTACTGTCC

SEQ ID NO 73 CrtR9 (reverse) 5 ' GGAATATTGCTGGTGATCCTTTCCA

SEQ ID NO 74 CrtRl 0 (reverse) 5 ' TTGCTGGTGATCCTTTCCATCCCTG

SEQ ID NO 75 CrtRl 1 (reverse) 5 ' CTACTCTGGAATATTGCTGGTGATCC

SEQ ID NO 76 CrtR12 (reverse) 5 ' CTTTTCTGGCAGCTGTATAGGCTGTACTG

SEQ ID NO 77 CrtR13 (reverse) 5 ' TCCTTTTCTGGCAGCTGTATAGGCTG

SEQ ID NO 78 CrtR14 (reverse) 5 ' CCCACTAACTTCTGTATATCATTGACAGTCCA

HIV-I PROTEASE PRIMERS

SEQ ID NO 79 CprM46LlA 5'AAACCTCCAATTCCTCCTATCAA

SEQ ID NO 80 CprM46LlG 5'AAACCTCCAATTCCTCCTATCAG

SEQ ID NO 81 CprM46MlT 5'AAACCTCCAATTCCTCCTATCAT

SEQ ID NO 82 CprM46I2T 5' GATAAAACCTCCAATTCCTCCTATT

SEQ ID NO 83 CprM46I2G 5' GATAAAACCTCCAATTCCTCCTATG

SEQ ID NO 84 CprM46I2A 5' GATAAAACCTCCAATTCCTCCTATA

SEQ ID NO 85 CprM46M2C 5' GATAAAACCTCCAATTCCTCCTATC

SEQ ID NO 86 CprI54VlC 5' TAAGTATTTGATCATACTGTCTTACTTTGAC

SEQ ID NO 87 CprI54IlT 5'TAAGTATTTGATCATACTGTCTTACTTTGAT

SEQ ID NO 88 CprA71AlG 5 ' CCTACTAATACTGTACCTATAG

SEQ ID NO 89 CprA71VlA 5 ' CCTACTAATACTGTACCTATAA SEQ ID NO 90 CprI84VlC 5' CTGAGTCAACATATTTCTTCCAATTAC

SEQ ID NO 91 CprI84IlT 5' CTGAGTCAACATATTTCTTCCAATTAT

SEQ ID NO 92 CprN88NlT 5 'ATCCAAGCTGAGTCAACATATT

SEQ ID NO 93 CprN88DlC 5 ΆTCCAAGCTGAGTCAACATATC

SEQ ID NO 94 CprN88SlA 5 'ATCCAAGCTGAGTCAACATATA

SEQ ID NO 95 CprN88N2T 5 ' CATCCAAGCTGAGTCAACATAT

SEQ ID NO 96 CprN88S2C 5 ' CATCCAAGCTGAGTCAACATAC

SEQ ID NO 97 CprN88S2G 5 ' CATCCAAGCTGAGTCAACATAG

SEQ ID NO 98 CprL90M2C 5 ' GTGTGCATCCAAGCTGAGTC

SEQ ID NO 99 CprL90L2A 5 ' GTGTGCATCCAAGCTGAGTA

SEQ ID NO 100 CprL90L2G 5 ' GTGTGCATCCAAGCTGAGTG

SEQ ID NO 101 CprL90L2T 5 ' GTGTGCATCCAAGCTGAGTT

SEQ ID NO 102 CprlFl (forward) 5 ΆCCCTTAACTTCCCTCAAATCACTCTTTGG

SEQ ID NO 103 CprRl (reverse) 5 ' CATTGTTTAACCTTTGGGCCATC

SEQ ID NO 104 CprR2 (reverse) 5 ' CATTGTTTAACCTTTGGGCCATCCATTCCT

SEQ ID NO 105 CprV32IlTΔ 5 ' GGCAAATTTATTTCTTCTAATAT

SEQ ID NO 106 CprV32VlCΔ 5 ' GGCAAATTTATTTCTTCTAATAC

SEQ ID NO 107 CprI50VlCΔ 5 ' TTACTTTGATAAAACCTCCAAC

SEQ ID NO 108 CprI50IlTΔ 5 ' TTACTTTGATAAAACCTCCAAT

SEQ ID NO 109 CprL90MlTΔ 5 ' TGCATCCAAGCTGAGTCAT

SEQ ID NO 110 CprL90LlAΔ 5 ' TGCATCCAAGCTGAGTCAA

SEQ ID NO 111 CprL90LlGΔ 5 ' TGCATCCAAGCTGAGTCAG

SEQ ID NO 112 CprD30DlC 5' GGCAAATTTATTTCTTCTAATACTGTATC

SEQ ID NO 113 CprD30NlT 5' GGCAAATTTATTTCTTCTAATACTGTATT

SEQ ID NO 114 CprV82VlC 5 ' TATTTCTTCCAATTATGTTGAC

SEQ ID NO 115 CprV82FlA 5 ' TATTTCTTCCAATTATGTTGAA

SEQ ID NO 116 CprV82IlT 5 ' TATTTCTTCCAATTATGTTGAT

SEQ ID NO 117 CprV82LlG 5 ' TATTTCTTCCAATTATGTTGAG

SEQ ID NO 118 CprV82V2A 5 'ATATTTCTTCCAATTATGTTGA

SEQ ID NO 119 CprV82A2G 5 'ATATTTCTTCCAATTATGTTGG

HIV-I gp41 primers

SEQ ID NO 120 Bgp41Q40QlT 5 ' CCCTCAGCAAATTGTTCTGCT SEQ ID NO 121 Bgp41Q40QlC 5 ' CCCTCAGCAAATTGTTCTGCC SEQ ID NO 122 Bgp41Q40HlA 5 ' CCCTCAGCAAATTGTTCTGCA SEQ ID NO 123 Bgp41Q40HlG 5 ' CCCTCAGCAAATTGTTCTGCG SEQ ID NO 124 Bgp41N42NlT 5 ' CAATAGCCCTCAGCAAATTGT SEQ ID NO 125 Bgp41N42TlG 5 ' CAATAGCCCTCAGCAAATTGG

SEQ ID NO 126 Bgp41N43NlT 5 ' GTTGCGCCTCAATAGCCCTCAGCAAATT

SEQ ID NO 127 Bgp41N43DlC 5 ' GTTGCGCCTCAATAGCCCTCAGCAAATC

SEQ ID NO 128 gp41Rin (reverse) 5 ' CACAGCCAGGACTCTTGCCTGGAG

SEQ ID NO 129 gp41Rin2 (reverse) 5'AAGCCTCCTACTATCATTATGAATATTTTTATATACCA

SEQ ID NO 130 gp41Fin ( forward) 5 ' GCAGCAGGAAGCACTATGGGCG

SEQ ID NO 131 gp41Fin3 (forward) 5 ' TGGGTTCTTGGGAGCAGCAGGAAG

SEQ ID NO 132 Bgp41G36GlC 5'AAATTGTTCTGCTGTTGCACTATAC

SEQ ID NO 133 Bgp41G36DlT 5'AAATTGTTCTGCTGTTGCACTATAT

SEQ ID NO 134 Bgp41V38VlCΔ 5 'AATTGTTCTGCTGTTGCAC

SEQ ID NO 135 Bgp41V38MlTΔ 5 'AATTGTTCTGCTGTTGCAT

SEQ ID NO 136 Bgp41V38V2AΔ 5 'AATTGTTCTGCTGTTGCA

SEQ ID NO 137 Bgp41V38A2GΔ 5 'AATTGTTCTGCTGTTGCG

SEQ ID NO 138 Bgp41V38E2TΔ 5 'AATTGTTCTGCTGTTGCT

SEQ ID NO 139 Bgp41Q39QlTΔ 5 ' GCAAATTGTTCTGCTGTT

SEQ ID NO 140 Bgp41Q39RlCΔ 5 ' GCAAATTGTTCTGCTGTC

SEQ ID NO 141 Bgp41V38VlC 5 ' CAAATTGTTCTGCTGTTGCAC

SEQ ID NO 142 Bgp41V38MlT 5 ' CAAATTGTTCTGCTGTTGCAT

SEQ ID NO 143 Bgp41V38V2A 5 ' GCAAATTGTTCTGCTGTTGCA

SEQ ID NO 144 Bgp41V38A2G 5 ' GCAAATTGTTCTGCTGTTGCG

SEQ ID NO 145 Bgp41V38E2T 5 ' GCAAATTGTTCTGCTGTTGCT

SEQ ID NO 146 Bgp41Q39QlT 5 ' CTCAGCAAATTGTTCTGCTGTT

SEQ ID NO 147 Bgp41Q39RlC 5 ' CTCAGCAAATTGTTCTGCTGTC

SEQ ID NO 148 Bgp41G36G2C 5'AATTGTTCTGCTGTTGCACTATACC

SEQ ID NO 149 Bgp41G36S2T 5'AATTGTTCTGCTGTTGCACTATACT

SEQ ID NO 150 Bgp41I37IlT 5' CAAATTGTTCTGCTGTTGCACTAT

SEQ ID NO 151 Bgp41I37VlC 5' CAAATTGTTCTGCTGTTGCACTAC

HIV-I Envelope V3 primers

SEQ ID NO 152 envV3subCfwd 5 'ACCATGCAATAATGTCAGCACAGTACAATGTACACATG SEQ ID NO 153 envV3subAlfwd 5 ' GCCATGCAAGAATGTCAGCACAGTACAATGCACACATG SEQ ID NO 154 envV3subBfwd 5 'ACCATGTACAAATGTCAGCACAGTACAATGTACACATG SEQ ID NO 155 envV3subDfwd 5 ' TCCATGCAAAAATGTCAGCACAGTACAGTGTACACATG SEQ ID NO 156 envV3subCrev 5 ' CAATTAAAGCTATGTGTTGTAATTTCTAGGTCCCCTCC SEQ ID NO 157 envV3subAlrev 5 ' CAATTAAAACTATGTGTTGTAATTTCTAGATCCCCTCC SEQ ID NO 158 envV3subBrev 5 ' CAATTAAAACTGTGCATTACAATTTCTGGGTCCCCTCC SEQ ID NO 159 envV3subDrev 5 ' CAATTAAAGCTGTGTGTTGTAATTTCTGGGTCCCCTCC Regions in bold indicate amino acid positions and mutations . Primers including a Δ symbol indicate alternative forms of primers .

HIV ARTIFICIAL GENE SEQUENCES

SEQ ID NO 160

>bcon_gp41 taggaaaagcaatgtatgcccctcccatcagaggacaaattagatgttcatcaaatattacagggctgctattaacaaga gatggtggtaataacgagaccgagatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataa atataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagag cagtgggaataggagctatgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacg gtacaggccagacaattattgtctggtatagtgcaacagcagaacaatttgctgagggctattgaggcgcaacagcatct gttgcaactcacagtctggggcatcaagcagctccaggcaagagtcctggctgtggaaagatacctaaaggatcaacagc tcctggggatttggggttgctctggaaaactcatttg

SEQ ID NO 161

>ccon_pol tgatgacagcatgtcagggagtgggaggacctagccacaaagcaagagtgttggctgaggcaatgagccaagcaaacaat acaaacataatgatgcagagaagcaattttaaaggccctaaaagaattgttaaatgtttcaactgtggcaaggaagggca catagccagaaattgcagggcccctaggaaaaaaggctgttggaaatgtggaaaggaaggacaccaaatgaaagactgta ctgagaggcaggctaattttttagggaaaatttggccttcccacaaggggaggccagggaatttccttcagaacagacca gagccaacagccccaccagcagagagcttcaggttcgaggagacaacccccgctccgaagcaggagccgaaagacaggga acccttaacttccctcaaatcactctttggcagcgaccccttgtctcaataaaagtagggggccagataaaggaggctct cttagacacaggagcagatgatacagtattagaagaaataaatttgccaggaaaatggaaaccaaaaatgataggaggaa ttggaggttttatcaaagtaagacagtatgatcaaatacttatagaaatttgtggaaaaaaggctataggtacagtatta gtaggacctacacctgtcaacataattggaagaaatatgttgactcagcttggatgcacactaaattttccaattagtcc cattgaaactgtaccagtaaaattaaagccaggaatggatggcccaaaggttaaacaatggccattgacagaagaaaaaa taaaagcattaacagcaatttgtgaagaaatggagaaggaaggaaaaattacaaaaattgggcctgaaaatccatataac actccagtatttgccataaaaaagaaggacagtactaagtggagaaaattagtagatttcagggaactcaataaaagaac tcaagacttttgggaagttcaattaggaataccacacccagcagggttaaaaaagaaaaaatcagtgacagtactggatg tgggggatgcatatttttcagttcctttagatgaaggcttcaggaaatatactgcattcaccatacctagtataaacaat gaaacaccagggattagatatcaatataatgtgcttccacagggatggaaaggatcaccagcaatattccagagtagcat gacaaaaatcttagagccctttagagcacaaaatccagaaatagtcatctatcaatatatggatgacttgtatgtaggat ctgacttagaaatagggcaacatagagcaaaaatagaggagttaagagaacatctattaaagtggggatttaccacacca gacaagaaacatcagaaagaacccccatttctttggatggggtatgaactccatcctgacaaatggacagtacagcctat acagctgccagaaaaggatagctggactgtcaatgatatacagaagttagtgggaaaattaaactgggcaagtcagattt acccagggattaaagtaaggcaactttgtaaactccttaggggggccaaagcactaacagacatagtaccactaactgaa gaagcagaattagaattggcagagaacagggaaattctaaaagaaccagtacatggagtatattatgacccatcaaaaga cttgatagctgaaatacagaaacaggggcatgaccaatggacatatcaaatttaccaagaacc

DETAILED DESCRIPTION OF THE INVENTION

The Applicants have, after much experimentation, developed a laboratory test to detect mutations associated with antiretroviral drug resistance in HIV-1 using an allele-specific real-time PCR assay. This method uses mutation-specific PCR primers designed to detect and quantify the subset of HIV-1 viruses carrying the nucleic acid changes which are associated with resistance. The invention provides primers, primer sets, and amplification conditions which offer increased sensitivity of detection, after having designed and tested many different primers, primer sets, and PCR conditions. In addition, the method of the invention can be used to estimate the relative proportion of resistant viruses in the total virus population in a host. This method is therefore suited for the detection and quantification of enfuvirtide, protease, and reverse transcriptase resistance mutations in HIV-1 and will provide more useful clinical information for patient management.

The following definitions and methods are provided to aid in defining the present invention and to guide persons having ordinary skill in the art in the interpretation of the present invention in a non-limiting way. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

The term "polynucleotide molecule", as used herein, refers to single- or double-stranded

DNA or RNA of genomic or synthetic origin, i.e., a polymer of deoxyribonucleotide or ribonucleotide bases, respectively, read from the 5' (upstream) end to the 3' (downstream) end. As used herein, the term "polynucleotide sequence" refers to the sequence of nucleotides or base pairs in a polynucleotide molecule.

As used herein, the term "substantially homologous" refers to polynucleotide molecules that demonstrate a substantial percent sequence identity with the promoters provided herein, wherein the polynucleotide molecules are useful in detecting virus populations or mutations in antiretroviral populations and have at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, or even greater sequence identity, such as 99% sequence identity with the polynucleotide sequences of the promoters described herein. As used herein, the term "percent sequence identity" refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference polynucleotide molecule (or its complementary strand) as compared to a test polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison).

As used herein, the term "homology" refers to the level of similarity or percent identity between polynucleotide sequences in terms of percent nucleotide positional identity, i.e., sequence similarity or identity. As used herein, the term homology also refers to the concept of similar functional properties among different polynucleotide molecules.

The term "stringent conditions" is functionally defined with regard to the hybridization of a nucleic-acid probe to a target nucleic acid. For applications requiring high selectivity, one will typically desire to employ relatively high stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCI at temperatures of about 5O⁰C to about 7O⁰C. A high stringency condition, for example, is to wash the hybridization filter at least twice with high-stringency wash buffer (0.2 x SSC, 0.1 % SDS, at 65°C). Appropriate moderate stringency conditions that promote DNA hybridization may be, for example, 6x sodium chloride/sodium citrate (SSC) at about 45⁰C, followed by a wash of 2 x SSC at 5O⁰C. Additionally, the salt concentration in the wash step can be selected from a low stringency of about 2 x SSC at 50⁰C to a high stringency of about 0.2 x SSC at 5O⁰C.

Protease and Reverse Transcriptase drug resistance testing

Methodology

Blood was collected in a 5 ml vacutainer tube. This was spun at 1000 rpm for 10 min to separate the plasma and buffy coat. One milliliter of plasma was collected and viral RNA extracted using an ABI 6100 machine and an extraction kit. The RNA was then converted to complementary DNA (reverse transcribed) in an ABI 7900 HT real-time PCR machine and pol sequences amplified using a set of gene-specific forward and reverse PCR primers (for example: prF1/prR1 ; rt1 F2/R7; and rt2F1/R5) with SYBR green RT-PCR master mix. This amplification product would also then serve as a suitable amplification product for the detection of the number of RNA particles using a standard curve method (viral load determination), shown in Figures 1 , 2 and 3.

Next, the amplified DNA was diluted in sterile water and aliquoted into a 384 well plate. An ABI7900HT machine was used to detect the wild-type and drug resistance mutations, using the primers as shown in the sequence listing and SYBRgreen PCR mastermix. The ΔCT method was used to calculate the proportion of wild-type and mutant virus populations and related to the viral load to calculate the number of mutant virus populations, as shown in Table 1 below. This also serves to assist clinicians in making informed decisions on treatment options for the patient.

Additionally, when PCR products are obtained by RT-PCR, but fail to generate resistance data in the protease or reverse transcriptase AS-PCR assay or if no known mutations are detected where resistance to protease or reverse transcriptase inhibitors is strongly suspected, the PCR products generated by RT-PCR can be used for DNA sequencing to obtain sequence data for protease and partial reverse transcriptase regions which can be analysed and resistance data provided within a 20% sensitivity range. This may be necessary when highly recombined or mutated HIV strains are encountered.

Results

Table 1 : Results of amplification efficiency and R² value for protease and reverse transcriptase wild-type primers and relative background detection levels (%) for associated protease and reverse transcriptase mutant primers tested for 4 log dilutions of plasmid positive control.

Determination of the levels of drug resistance within patients on antiretroviral therapy is an important part of the treatment regimen as drug resistance will influence the choice of drugs as well as efficacy of drugs being prescribed. The development of a more sensitive drug resistance test can assist clinicians in tracking the development of drug resistance to certain drugs and over time make informed decisions on when to switch patients to more efficient drugs.

Enfuvirtide resistance testing

Methodology

Blood was collected in a 5 ml vacutainer tube. This was spun at 1000 rpm for 10 min to separate the plasma and buffy coat. One milliliter of plasma was collected and viral RNA extracted using an ABI 6100 machine and extraction kit. The RNA was then converted to complementary DNA (reverse transcribed) in an ABI7900HT real-time PCR machine using reverse transcriptase and amplified using a set of gene-specific forward and reverse PCR primers (for example: Fin3/Rin) with SYBRgreen RT-PCR master mix. This amplification would then also serve as a suitable amplification product for the detection of the number of RNA particles using a standard curve method (viral load determination). Next the amplified DNA was used during allele specific PCR to detect wild-type and drug resistant mutated populations with SYBRgreen PCR mastermix. The ΔCT method was used to calculate the proportion of wild-type and mutant virus populations and related to the viral load to calculate the number of mutant virus populations and help clinicians make informed decisions on treatment options for the patient.

Additionally, failure to generate resistance data in the gp41 HR1 AS-PCR assay, or if no known mutations are detected where resistance to enfuvirtide is strongly suspected, the PCR product generated by primer set gp41 Fin/gp41 Rin2 can be used for DNA sequencing to obtain sequence data for both the HR1 and HR2 region of gp41. Furthermore, compensatory mutations can develop in the V3 region of the envelope gene. In order to determine changes over time, the V3 region can be DNA sequenced using the V3 primers described.

Results

Table 2: Results of amplification efficiency and R² value of gp41 wild-type primers and background detection levels (%) for associated gp41 mutant primers tested for 4 log dilutions of plasmid positive control.

Using allele specific real-time PCR with the primers of the invention to detect mutations in the gp41 region of the envelope gene (i.e. to detect possible enfuvirtide resistance) of HIV-1 is a suitable method of determining viral load and quantifying viral mutants, as shown by the results presented in Table 2. This also assists in the calculation of proportional virus populations, both wild-type and mutant, and assists clinicians in making more informed decisions regarding treatment options.

The Applicants are of the opinion that they have invented a quick and easy method, primers, and primer sets for both detecting and quantifying nucleic acid changes in HIV-1 samples, using real-time allele-specific PCR (AS-PCR). This method uses mutation and wild-type specific DNA probes (PCR primers) designed to specifically detect and quantify the subset of HIV-1 viruses carrying the DNA changes in viral genes which may be associated with drug resistance. The sensitivity of detection is greater than can be achieved by DNA sequencing methods (<20%) known to the inventors and, in addition, the test can estimate the relative proportion of resistant viruses in the total population. The technique of the invention can also be used to obtain DNA sequence data from the PCR products generated by RT-PCR should the AS-PCR assay fail for any reason and hence still supply resistance data within a 20% sensitivity range. Coupled to a viral load measurement the absolute number of resistant viruses can be calculated and this serves as an early warning system for when drug resistant viruses approach critical levels which may compromise patient health and allow transmission of resistant viruses. Accordingly, the treatment regimen can then be changed and cross-resistant interactions can be avoided.

This method is therefore ideally suited for the detection and quantification of antiretroviral drug-resistance mutations in the HIV-1 reverse transcriptase, HIV-1 protease genes and HIV-1 gp41 HR1 gene and will provide more informative information for patient clinical management.

Claims

1. A method of screening for mutations associated with resistance in a viral population to any one or more antiretroviral drugs selected from the group comprising abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, zidovudine, delavirdine, efavirenz and nevirapine, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence selected from any one or more of SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and screening the amplification product for the presence of mutations associated with resistance to any one or more of such antiretroviral drugs.

2. The method as claimed in claim 1 , in which the mutations are any one or more of the HIV-1 protease mutations shown in Table 1.

3. The method as claimed in claim 1 or claim 2, in which screening for mutations associated with resistance to any or all of the abovementioned drugs occurs substantially simultaneously in a real-time PCR instrument.

4. The method as claimed in any one of claims 1 to 3, in which detecting the levels of a virus population having one or more mutations includes the step of quantifying the relative levels of amplification product using semi-quantitative PCR.

5. The method as claimed in claim 4, in which the semi-quantitative PCR is done in a real-time PCR instrument.

6. The method as claimed in any one of claims 1 to 5, in which viral RNA is extracted from the virus populations obtained from blood specimens from infected hosts and converted to DNA in a real-time PCR instrument using the primers, prior to further amplification.

7. The method as claimed in claim 6, which includes the step of utilizing the DNA product for the detection of the absolute number of RNA particles using a standard curve method.

8. The method as claimed in any one of claims 1 to 7, which includes the step of quantifying the level of resistant viruses to wild-type viruses in the host by ΔCT testing.

9. The method as claimed in any one of claims 1 to 8, which is performed in an allele specific manner.

10. The method as claimed in any one of claims 1 to 9, which is performed in a 96 or

384 well plate.

1 1. The method as claimed in any one of claims 1 to 10, in which the proportion of wild- type to mutant viruses is related back to a viral load value to calculate the absolute number of mutant or wild-type viruses in a total viral population in a host.

12. A method of screening for mutations associated with resistance in a viral population to any one or more antiretroviral drugs selected from the group comprising atazanavir, fosamprenavir, darunavir, indinavir, lopinavir, nelfinavir, saquinavir, tiprannavir and ritonavir, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and screening the amplification product for the presence of mutations associated with resistance to any one or more of such antiretroviral drugs.

13. The method as claimed in claim 12, in which the mutations are any one or more of the HIV-1 reverse transcriptase mutations shown in Table 1.

14. The method as claimed in claim 12 or claim 13, in which screening for mutations associated with resistance to any or all of the abovementioned drugs occurs substantially simultaneously in a real-time PCR instrument.

15. The method as claimed in any one of claims 12 to 14, in which detecting the levels of a virus population having one or more mutations includes the step of quantifying the relative levels of amplification product using semi-quantitative PCR.

16. The method as claimed in claim 15, in which the semi-quantitative PCR is done in a real-time PCR instrument.

17. The method as claimed in any one of claims 12 to 16, in which viral RNA is extracted from the virus populations obtained from blood specimens from infected hosts and converted to DNA in a real-time PCR instrument using the primers, prior to further amplification.

18. The method as claimed in claim 17, which includes the step of utilizing the DNA product for the detection of the absolute number of RNA particles using a standard curve method.

19. The method as claimed in any one of claims 12 to 18, which includes the step of quantifying the level of resistant viruses to wild-type viruses in the host by ΔCT testing.

20. The method as claimed in any one of claims 12 to 19, which is performed in an allele specific manner.

21. The method as claimed in any one of claims 12 to 20, which is performed in a 96 or

384 well plate.

22. The method as claimed in any one of claims 12 to 21 , in which the proportion of wild- type to mutant viruses is related back to a viral load value to calculate the absolute number of mutant or wild-type viruses in a total viral population in a host.

23. A method of screening for mutations associated with resistance in a viral population to enfuvirtide, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and screening the amplification product for the presence of mutations associated with resistance to any one or more of such antiretroviral drugs.

24. The method as claimed in claim 23, in which the mutations are any one or more of the HIV-1 gp41 mutations shown in Table 2.

25. The method as claimed in claim 23 or claim 24, in which screening for mutations associated with resistance to any or all of the abovementioned drugs occurs substantially simultaneously in a real-time PCR instrument.

26. The method as claimed in any one of claims 23 to 25, in which detecting the levels of a virus population having one or more mutations includes the step of quantifying the relative levels of amplification product using semi-quantitative PCR.

27. The method as claimed in claim 26, in which the semi-quantitative PCR is done in a real-time PCR instrument.

28. The method as claimed in any one of claims 23 to 27, in which viral RNA is extracted from the virus populations obtained from blood specimens from infected hosts and converted to DNA in a real-time PCR instrument using the primers, prior to further amplification.

29. The method as claimed in claim 28, which includes the step of utilizing the DNA product for the detection of the absolute number of RNA particles using a standard curve method.

30. The method as claimed in any one of claims 23 to 29, which includes the step of quantifying the level of resistant viruses to wild-type viruses in the host by ΔCT testing.

31. The method as claimed in any one of claims 23 to 30, which is performed in an allele specific manner.

32. The method as claimed in any one of claims 23 to 31 , which is performed in a 96 or 384 well plate.

33. The method as claimed in any one of claims 23 to 32, in which the proportion of wild- type to mutant viruses is related back to a viral load value to calculate the absolute number of mutant or wild-type viruses in a total viral population in a host.

34. A method for quantifying the levels of a virus population in a specimen, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence selected from any one or more of SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; or amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; or amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; or amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 152 to 155, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 156 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and subjecting the results of the polymerase chain reaction to ΔCT testing.

35. The method as claimed in claim 34, in which quantification of levels of viral populations occurs substantially simultaneously in a real-time PCR instrument using semiquantitative PCR.

36. The method as claimed in claim 34 or 35, in which viral RNA is extracted from the virus populations obtained from blood specimens from infected hosts and converted to DNA in a real-time PCR instrument using the primers, prior to further amplification.

37. The method as claimed in claim 36, which includes the step of utilizing the DNA product for the detection of the absolute number of RNA particles using a standard curve method.

38. The method as claimed in any one of claims 34 to 37, which is performed in a 96 or 384 well plate.

39. The method as claimed in any one of claims 34 to 38, in which the proportion of wild- type to mutant viruses is related back to a viral load value to calculate the absolute number of mutant or wild-type viruses in a total viral population in a host.

40. A method of detecting the presence of a retrovirus, the method including the steps of: extracting nucleic acid from specimens obtained from potentially infected hosts; and amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence selected from any one or more of SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; or amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; or amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; or amplifying a nucleic acid product by polymerase chain reaction using at least one forward primer having a sequence of SEQ ID NO 152 to 155, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 156 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and screening the amplification product to determine whether a retrovirus is present in the sample.

41. The method as claimed in claim 40, in which screening for the presence of a retrovirus occurs in a real-time PCR instrument.

42. The method as claimed in claim 40 or 41 , in which viral RNA is extracted from the virus populations obtained from blood specimens from infected hosts and converted to DNA in a real-time PCR instrument using the primers, prior to further amplification.

43. The method as claimed in claim 42, which includes the step of utilizing the DNA product for the detection of the absolute number of RNA particles using a standard curve method.

44. The method as claimed in any one of claims 40 to 43, which includes the step of quantifying the level of resistant viruses to wild-type viruses in the host by ΔCT testing.

45. The method as claimed in any one of claims 40 to 44, which is performed in an allele specific manner.

46. The method as claimed in any one of claims 40 to 45, which is performed in a 96 or 384 well plate.

47. The method as claimed in any one of claims 40 to 46, in which the proportion of wild- type to mutant viruses is related back to a viral load value to calculate the absolute number of mutant or wild-type viruses in a total viral population in a host.

48. An oligonucleotide primer having a sequence selected from the group including any one of SEQ ID NO 1 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; substitution, deletion, addition or insertion variants thereof.

49. A set of oligonucleotide primers, wherein the set includes at least one oligonucleotide primer having a sequence selected from any one of SEQ ID NO 1 to 159 inclusive; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

50. The set of oligonucleotide primers as claimed in claim 49, wherein the set includes at least one forward primer having a sequence selected from SEQ ID NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

51. The set of oligonucleotide primers as claimed in claim 49, wherein the set includes at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

52. The set of oligonucleotide primers as claimed in claim 49, wherein the set includes at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID NO 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

53. The set of oligonucleotide primers as claimed in claim 49, wherein the set includes at least one forward primer having a sequence of SEQ ID NO 152 to 155, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 156 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

54. The oligonucleotide primer as claimed in claim 48, which contains modified bases.

55. The oligonucleotide primer as claimed in claim 54, which is modified to contain Locked Nucleic Acid (LNA) modifications at the 3' end thereof.

56. A kit for the detection of mutations associated with antiretroviral drug resistance in a virus population present in a host, the kit including: at least one forward primer having a sequence selected from any one or more of SEQ ID

NO 40, 41 or SEQ ID NO 59 to 65, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 1 to 39 or SEQ ID NO 42 to 58 or SEQ ID NO 66 to 78; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and/or at least one forward primer having a sequence of SEQ ID NO 102, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 79 to 101 or SEQ ID NO 103 to 119; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and/or at least one forward primer having a sequence of SEQ ID NO 130 or 131 , and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 120 to 129 or SEQ ID 132 to 151 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof; and/or at least one forward primer having a sequence of SEQ ID NO 152 to 155, and at least one reverse primer having a sequence selected from any one or more of SEQ ID NO 156 to 159; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

57. The kit as claimed in claim 56, which includes extraction means for the extraction of nucleic acid from specimens obtained from potentially diseased hosts.

58. An artificial HIV-1 nucleic acid sequence, the nucleic acid sequence having homology to any one of SEQ ID NO 160 or SEQ ID NO 161 ; parts thereof; sequences complementary thereto; sequences hybridizing under stringent conditions thereto; and substitution, deletion, addition or insertion variants thereof.

59. Use of the primers or primer sets of any one of claims 48 to 55, for the detections of any one or more of the following protease mutations in HIV-1 populations in a host: D30N, V32I, M46L/I, I50V, I54V, A71V, I84V, N88D/S, and L90M.

60. Use of the primers or primer sets of any one of claims 48 to 55, for the detections of any one or more of the following reverse transcriptase mutations in HIV-1 populations in a host:

M41 L, K65R, D67N, T69S/A/N, K70R/E, K103N, V106M/A, Y181 C, M184V, Y188C/L, G190A, L210W, T215S/I/Y, K219Q, and M230L.

61. Use of the primers or primer sets of any one of claims 48 to 55, for the detections of any one or more of the following gp41 mutations in HIV-1 populations in a host: G36D/S, I37V, V38M/A/E, Q39R, Q40H, N42T and N43D.

62. A method as claimed in any one of claims 1 , 12, 23, 34 or 40, substantially as herein described and illustrated.

63. A primer as claimed in claim 48, substantially as herein described and illustrated.

64. A set of primers as claimed in claim 49, substantially as herein described and illustrated.

65. A kit as claimed in claim 56, substantially as herein described and illustrated.

66. An artificial sequence as claimed in claim 58, substantially as herein described and illustrated.

67. Use of the primers or primer sets as claimed in any one of claims 59, 60, or 61 , substantially as herein described and illustrated.