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 determining the presence or absence of HBV genotype A in a biological sample, comprising:
(i) providing a biological sample comprising polynucleic acids;
(ii) optionally releasing, isolating and/or concentrating the polynucleic acids present in the sample;
(iii) optionally amplifying the HBsAg region, or part thereof, of the HBV gene present in said sample with at least one primer pair;
(iv) hybridizing the polynucleic acids of step (i) or (ii) or (iii) with at least one nucleotide probe selected from the group consisting of SEQ ID NO: 77, SEQ ID NO: 140, SEQ ID NO: 193, the complement of SEQ ID NO:77, the complement of SEQ ID NO:140, and the complement of SEQ ID NO:193;
(v) detecting the hybrid(s) formed in step (iv);
(vi) inferring the presence of absence of HBV genotype A in said sample from the hybridization signal(s) obtained in step (v).
2. The method according to claim 1 , for further determining the presence or absence of HBV genotype B in the biological sample, whereby step (iv) further comprises hybridizing the polynucleic acids of step (i) or (ii) or (iii) with at least one nucleotide probe selected from the group consisting of SEQ ID NOs:78, 142-147, 149 and their complement sequences.
3. The method according to claim 1 , for further determining the presence or absence of HBV genotype C in the biological sample, whereby step (iv) further comprises hybridizing the polynucleic acid of step (i) or (ii) or (iii) with at least one nucleotide probe selected from the group consisting of SEQ ID NOs: 79, 150-161, 204 and their complement sequences.
4. The method according to claim 1 , for further determining the presence or absence of HBV genotype D in the biological sample, whereby step (iv) further comprises hybridizing the polynucleic acids of step (i) or (ii) or (iii) with at least one nucleotide probe selected from the group consisting of SEQ ID NOs: 82, 162-169, 208 and their complement sequences.
5. The method according to claim 1 , for further determining the presence or absence of HBV genotype E in the biological sample, whereby step (iv) further comprises hybridizing the polynucleic acids of step (i) or (ii) or (iii) with at least one nucleotide probe selected from the group consisting of SEQ ID NOs: 170-176, 213, and their complement sequences.
6. The method according to claim 1 , for further determining the presence or absence of HBV genotype F in the biological sample, whereby step (iv) further comprises hybridizing the polynucleic acids of step (i) or (ii) or (iii) with at least one nucleotide probe selected from the group consisting of SEQ ID NOs:178-189, 214-219, and their complement sequences.
7. The method according to claim 1 wherein the primers of said at least one primer pair of step (iii) are selected from the group consisting of SEQ ID NOs: 75-76, 94, 104, 105, 112 and 134-135.
8. The method according to claim 1 wherein step (iv) of hybridizing the polynucleic acids of step (i) or (ii) or (iii) with at least one nucleotide probe is a reverse hybridization step.