Real-time PCR point mutation assays for detecting HIV-1 resistance to antiviral drugs

A method for detecting a mutation in a nucleic acid sequence relating to a HIV-1 protein of reverse transcriptase or protease in a subject, comprising:

• contacting a DNA molecule derived from an HIV-virus and encoding the HIV-1 protein with a common primer pair comprising a forward primer and a reverse primer and a mutation-specific primer pair comprising at least one forward primer and at least one reverse primer, in an amplification reaction;
• wherein the common primer pair generates a polymerase-chain-reaction (PCR) population of a common amplicon;
• and wherein the mutation-specific primer pair generates a PCR population of a mutation-specific amplicon;
• and
• detecting said mutation by comparing the differential amplifications of said common amplicon and said mutation-specific amplicon;
• wherein the mutation relating to the HIV-1 protein of protease is selected from the group consisting of 30N and 90M;
• and
• wherein the mutation-specific forward primer for detecting the mutation relating to the HIV-1 protein of protease is selected from the group consisting of SEQ ID NOs:
• 48, 49, 53, 54, 55, 78, 79, and 80.

A method for detecting a mutation in a nucleic acid sequence relating to a HIV-1 protein of reverse transcriptase or protease in a subject, comprising:

• contacting a DNA molecule derived from an HIV-virus and encoding the HIV-1 protein with a common primer pair comprising a forward primer and a reverse primer and a mutation-specific primer pair comprising at least one forward primer and at least one reverse primer, in an amplification reaction;
• wherein the common primer pair generates a polymerase-chain-reaction (PCR) population of a common amplicon;
• and wherein the mutation-specific primer pair generates a PCR population of a mutation-specific amplicon;
• and
• detecting said mutation by comparing the differential amplifications of said common amplicon and said mutation-specific amplicon, and
• wherein the mutation-specific reverse primer for detecting the mutation relating to the HIV-1 protein of reverse transcriptase is selected from the group consisting of SEQ ID NOs:
• 26, 36, 66, 11, 8, 14, 20, 72, 73, 30, 85, and 45.

A method for detecting a mutation in a nucleic acid sequence relating to a HIV-1 protein of reverse transcriptase or protease in a subject, comprising:

• contacting a DNA molecule derived from an HIV-virus and encoding the HIV-1 protein with a common primer pair comprising a forward primer and a reverse primer and a mutation-specific primer pair comprising at least one forward primer and at least one reverse primer, in an amplification reaction;
• wherein the common primer pair generates a polymerase-chain-reaction (PCR) population of a common amplicon;
• and wherein the mutation-specific primer pair generates a PCR population of a mutation-specific amplicon;
• and
• detecting said mutation by comparing the differential amplifications of said common amplicon and said mutation-specific amplicon, and
• wherein the mutation-specific reverse primer for detecting the mutation relating to the HIV-1 protein of protease is selected from the group consisting of SEQ ID NOs:
• 50, 56, and 81.

A method for detecting a mutation in a nucleic acid sequence relating to a HIV-1 protein of reverse transcriptase or protease in a subject, comprising:

• contacting a DNA molecule derived from an HIV-virus and encoding the HIV-1 protein with a common primer pair comprising a forward primer and a reverse primer and a mutation-specific primer pair comprising at least one forward primer and at least one reverse primer, in an amplification reaction;
• wherein the common primer pair generates a polymerase-chain-reaction (PCR) population of a common amplicon;
• and wherein the mutation-specific primer pair generates a PCR population of a mutation- specific amplicon;
• and
• detecting said mutation by comparing the differential amplifications of said common amplicon and said mutation-specific amplicon, wherein the PCR population of the common amplicon is further contacted by a probe selected from the group consisting of SEQ ID NOs:
• 9, 86, 87, 67, 68, 32, 47, 76, 77, 52, 58, 99 and 82.

A method for detecting a mutation in a nucleic acid sequence relating to a HIV-1 protein of reverse transcriptase or protease in a subject, comprising:

• contacting a DNA molecule derived from an HIV-virus and encoding the HIV-1 protein with a common primer pair comprising a forward primer and a reverse primer and a mutation-specific primer pair comprising at least one forward primer and at least one reverse primer, in an amplification reaction;
• wherein the common primer pair generates a polymerase-chain-reaction (PCR) population of a common amplicon;
• and wherein the mutation-specific primer pair generates a PCR population of a mutation-specific amplicon;
• and
• detecting said mutation by comparing the differential amplifications of said common amplicon and said mutation-specific amplicon, wherein the common forward primer is SEQ ID NO:
• 7.

A method for detecting a mutation in a nucleic acid sequence relating to a HIV-1 protein of reverse transcriptase or protease in a subject, comprising:

• contacting a DNA molecule derived from an HIV-virus and encoding the HIV-1 protein with a common primer pair comprising a forward primer and a reverse primer and a mutation-specific primer pair comprising at least one forward primer and at least one reverse primer, in an amplification reaction;
• wherein the common primer pair generates a polymerase-chain-reaction (PCR) population of a common amplicon;
• and wherein the mutation-specific primer pair generates a PCR population of a mutation-specific amplicon;
• and
• detecting said mutation by comparing the differential amplifications of said common amplicon and said mutation-specific amplicon, wherein the common reverse primer is SEQ ID NO:
• 8.