HIV-1 genotyping assay for global surveillance of HIV-1 drug resistance

A method of genotyping HIV-1 in a sample, comprising:

• (a) contacting a sample obtained from a subject with a first forward nucleic acid primer comprising a mixture of SEQ ID NO:
• 1 and 2 and a first reverse nucleic acid primer consisting of SEQ ID NO:
• 4, or contacting the sample obtained from a subject with a first forward nucleic acid primer consisting of SEQ ID NO:
• 3 and a first reverse nucleic acid primer consisting of SEQ ID NO:
• 4, thereby generating a first reaction mixture;
• (b) incubating the first reaction mixture under conditions sufficient to amplify by polymerase chain reaction (PCR) a portion of HIV-1 pol in the first reaction mixture, thereby generating a first amplification product;
• (c) contacting the first amplification product with a second forward nucleic acid primer consisting of SEQ ID NO:
• 5 and a second reverse nucleic acid primer consisting of SEQ ID NO:
• 6, thereby generating a second reaction mixture;
• (d) incubating the second reaction mixture under conditions sufficient to amplify by PCR a portion of HIV-1 pol comprising HIV-1 reverse transcriptase (RT) and HIV-1 protease (PR) in the second reaction mixture, thereby generating a second amplification product;
• and
• (e) sequencing the first amplification product and the second amplification product to determine the genotype of HIV-1, or subjecting the first amplification product and the second amplification product to allele-specific analysis to determine the genotype of HIV-1, wherein the method can detect a viral load of ≧1,000 copies/ml.