Method for typing and detecting HBV
The present invention relates to a method for detection and/or genetic analysis of HBV in a biological sample, comprising hybridizing the polynucleic acids of the sample with a combination of at least two nucleotide probes, with said combination hybridizing specifically to a mutant target sequence chosen from the HBV RT pol gene region and/or to a mutant target sequence chosen from the HBV preCore region and/or to a mutant target sequence chosen from the HBsAg region of HBV and/or to a HBV genotype-specific target sequence, with said target sequences being chosen from FIG. 1 , and with said probes being applied to known locations on a solid support and with said probes being capable of hybridizing to the polynucleic acids of the sample under the same hybridization and wash conditions, or with said probes hybridizing specifically with a sequence complementary to any of said target sequences, or a sequence where T of said target sequence is replaced by U; and detecting the hybrids formed; and inferring the HBV genotype and/or mutants present in said sample from the differential hybridization signal(s) obtained. The invention further relates to sets of nucleotide probes and possibly primers useful in said methods as well as to their use in a method for typing and/or detecting HBV and to assay kits using the same.
1. A method for identifying a HBV strain resistant to lamivudine and penciclovir in a sample of serum of a HBV-infected patient, comprising:
(i) amplifying at least a part of the HBV gene present in said sample with at least one primer pair;
(ii) hybridizing the polynucleic acid products of the amplification of (i) with at least one probe which hybridizes specifically under stringent conditions to the codon coding for V of the YVDD motif of the HBV RT pol gene target sequence comprising the codon coding V of the YVDD motif;
wherein said probe hybridizing specifically under stringent conditions to a HBV RT pol gene target sequence comprising the codon coding for the V of the YVDD motif is selected from the group consisting of SEQ ID NOs: 101-103, 116, 117, 130-133, 244-248, 251-260 and 262-266, or wherein said probe hybridizes specifically under stringent conditions to a complement of a sequence selected from the group consisting of 101-103, 116, 117, 130-133, 244-248, 251-260, and 262-266;
(iii) based on the results of hybridizing (ii), determining the presence or absence of a mutation in the HBV RT pol gene resulting in an amino acid change in the YMDD motif of the HBV polymerase wherein said amino acid change is M to V in the YMDD motif and thereby identify the existence of a HBV strain resistant to lamivudine and penciclovir in said sample.
2. A method according to claim 1 wherein one of the primers of the at least one primer pair is selected from the group consisting of SEQ ID NOs: 75, 94, 104, 105, 134 and 135.