Establishment of a PCR-product direct sequencing for the detection of HBV YMDD mutation

Abstract

Objective To develop an assay of PCR-produet direct sequencing to detect hepatitis B virus (HBV) YMDD mutation, and compare the results gained by the sequencing and traditional real-time fluorescent PCR assays. Methods Serum samples were collected from 103 patients with chronic hepatitis B. HBV DNA were extracted from sers. YMDD mutation was detected by a commercial real-time PCR assay. Meanwhile, HBV reverse transcriptase-encoding gene was amplified by a nested PCR assay. The PCR products were directly subjected to sequencing at two directions, and the sequencing results were analyzed by NTI program. Using Kappa test, comparison was made between the results of rtM204-site mutations obtained by the direct sequencing and YMDD mutations by the real-time fluorescent PCR. Results The direct sequencing assay proved to be highly effective with bread range of detection in viral load from 500 to 1010copies/ml. And it may simultaneously avoid inhibitory effect caused by high viral load. The coincidence rates between two assays were 100% for YIDD, 97. 1% for YVDD, 76. 2% for YIDD/YVDD coexistence (Kappa = 0. 853, P < 0. 01). Conclusions The direct sequencing assay for HBV drug-resistant mutation detection is highly sensitive with broad dynamic range. It has high coincidence rate with real-time fluorescent PCR assay with advantage of detecting YMDD, YIDD and YVDD mutations simultaneously. Key words: Hepatitis B virus; Sequence analysis, DNA; Polymerase chain reaction; Mutation