Multiplex locked nucleic acid probes for analysis of hepatitis B virus mutants using real-time PCR.
Current methods of detecting hepatitis B virus (HBV) mutations are time consuming, labor intensive, and not suitable for screening large numbers of samples. A multiplex real-time PCR approach presented in this article is a hepatitis B virus quantification method that employs the SYBR Green I dye in conjunction with wild-type HBV sequence-specific locked nucleic acid (LNA) probes. The three short LNA probes distinguished the wild-type strain or three groups of mutants (rt173, rt180/rt181, and rt202/rt204) depending on perfect-match hybrids or mismatch within one template simultaneously. Primers labeled with quencher minimized the background signals. This sensitive approach could quantify 10(2) copies of HBV virus, and as low as 1% mutants among 10(4) copies of wild-type HBV could be identified. The technique is handy and convenient, requiring only 3.5 h to analyze 30 hepatitis B surface antigen-positive serum samples. The HBV isolates were confirmed by direct sequencing. Our data indicate that real-time PCR with SYBR Green I dye is a reliable, rapid, and convenient technique for HBV quantification. Furthermore, by incorporating fluorescent LNA probes, this technique becomes handy in identifying and classifying mutations in the HBV polymerase gene. Being sensitive, specific, accurate, rapid, and convenient in nature, this technique could be a suitable diagnostic tool with wide application particularly in cases in which large volumes of clinical samples are handled.
Sun
Zhen
Z
Institute of Infectious Disease, First Affiliated Hospital, Medical College, Zhejiang University, and Key Laboratory of Infectious Disease, Ministry of Public Health of China, Hangzhou, Zhejiang 310016, Peoples' Republic of China.
Zhou
Linfu
L
Zeng
Haiyan
H
Chen
Zhi
Z
Zhu
Haihong
H
D001483:Base Sequence; D052160:Benzothiazoles; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000235:genetics; D003959:Diamines; D005456:Fluorescent Dyes; D005814:Genes, Viral; D006509:Hepatitis B / Q000821:virology; D006514:Hepatitis B Surface Antigens / Q000097:blood / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods; D011804:Quinolines
D052160:Benzothiazoles; D004279:DNA, Viral; D003959:Diamines; D005456:Fluorescent Dyes; D006514:Hepatitis B Surface Antigens; D015345:Oligonucleotide Probes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I