US 6,713,251 B2 · Method for detection of drug-induced mutations in the reverse transcriptase gene

Abstract

The present invention relates to a method for the rapid and reliable detection of drug-induced mutations in the reverse transcriptase gene allowing the simultaneous characterization of a range of codons involved in drug resistance using specific sets of probes optimized to function together in a reverse-hybridization assay. More particularly, the present invention relates to a method for determining the susceptibility to antiviral drugs of HIV strains present in a biological sample, comprising: (i) if need be releasing, isolating or concentrating the polynucleic acids present in the sample; (ii) if need be amplifying the relevant part of the reverse transcriptase genes present in said sample with at least one suitable primer pair; (iii) hybridizing the polynucleic acids of step (i) or (ii) with at least two RT gene probes hybridizing specifically to one or more target sequences with said probes being applied to known locations on a solid support and with said probes being capable of simultaneously hybridizing to their respective target regions under appropriate hybridization and wash conditions allowing the detection of homologous targets, or said probes hybridizing specifically with a sequence complementary to any of said target sequences, or a sequence wherein T is replaced by U; (iv) detecting the hybrids formed in step (iii); (v) inferring the nucleotide sequence at the codons of interest and/or the amino acids of the codons of interest and/or antiviral drug resistance spectrum, and possibly the type of HIV isolates involved from the differential hybridization signal(s) obtained in step (iv).

Claims23

Dependent claims

A kit for inferring the nucleotide sequence at codons of interest in the HIV reverse transciptase (RT) and/or the amino acids corresponding to these codons and/or the HIV RT resistance spectrum of HIV isolates present in a biological sample comprising:

(i) optionally, a means for releasing, isolating, or concentrating the polynucleic acids present in said sample;
(ii) optionally, at least one suitable set of primers;
(iii) at least two different probes, wherein each probe is capable of hybridizing specifically to one or more target codons within any region I to VIII as represented in FIG. 1, said probes optionally fixed to a solid support;
(iv) a hybridization buffer, or components necessary for producing said buffer;
(v) a wash solution, or components necessary for producing said solution;
(vi) optionally, a means for detecting the hybrids resulting from the preceding hybridization;
(vii) optionally, a means for attaching said probe to a solid support wherein the set of primers is selected from the group consisting of:
SEQ ID No:
162 and 163, and
SEQ ID No:
164 and 39.

8 more elements

A kit for inferring the nucleotide sequence at codons of interest in the HIV RT gene and/or the amino acids corresponding to these codons and/or the antiviral drug resistance spectrum of HIV isolates present in a biological sample, the kit comprising the following components:

at least two different probes, wherein the probes are selected from the group consisting of SEQ ID NO:
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 37, 40, 41, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 114, 115, 117, 118, 119, 120, 121, 122, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 154, 155, 156, 157 and 159;
a hybridization buffer, or components necessary for producing said buffer;
and
a wash solution, or components necessary for producing said solution.

2 more elements

Dependencies are inferred from the claim text.

Method for detection of drug-induced mutations in the reverse transcriptase gene

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Method for detection of drug-induced mutations in the reverse transcriptase gene

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Abstract

Claims23