Establishment of real time allele specific locked nucleic acid quantitative pcr for detection of HBV YIDD (ATT) mutation and evaluation of its application

Abstract

Background: Long-term use of nucleos(t)ide analogues can increase risk of HBV drug-resistance mutations. The rtM204I (ATT coding for isoleucine) is one of the most important resistance mutation sites. Establishing a simple, rapid, reliable and highly sensitive assay to detect the resistant mutants as early as possible is of great clinical significance. Methods: Recombinant plasmids for HBV YMDD (tyrosine-methionine-aspartate-aspartate) and YIDD (tyrosine- isoleucineaspartate- aspartate) were constructed by TA cloning. Real time allele specific locked nucleic acid quantitative PCR (RT-ASLNA- qPCR) with SYBR Green I was established by LNA-modified primers and evaluated with standard recombinant plasmids, clinical templates (the clinical wild type and mutant HBV DNA mixture) and 102 serum samples from nucleos(t)ide analogues-experienced patients. The serum samples from a chronic hepatitis B (CHB) patient firstly received LMV mono therapy and then switched to LMV + ADV combined therapy were also dynamically analyzed for 10 times. Results: The linear range of the assay was between 16109 copies/ml and 16102 copies/ml. The low detection limit was 16101 copies/ml. Sensitivity of the assay were 1026, 1024 and 1022 in the wild-type background of 16109 copies/ml, 16107 copies/ml and 16105 copies/ml, respectively. The sensitivity of the assay in detection of clinical samples was 0.03%. The complete coincidence rate between RT-AS-LNA-qPCR and direct sequencing was 91.2% (93/102), partial coincidence rate was 8.8% (9/102), and no complete discordance was observed. The two assays showed a high concordance (Kappa = 0.676, P = 0.000). Minor variants can be detected 18 weeks earlier than the rebound of HBV DNA load and alanine aminotransferase level. Conclusions: A rapid, cost-effective, high sensitive, specific and reliable method of RT-AS-LNA-qPCR with SYBR Green I for early and absolute quantification of HBV YIDD (ATT coding for isoleucine) variants was established, which can provide valuable information for clinical antiretroviral regimens. © 2014 Zeng et al.