High resolution melting analysis with unlabeled probe for detection of rt204 mutation in HBV P gene / 中华临床感染病杂志

ABSTRACT

Objective To establish a method of high resolution melting analysis (HRM) with unlabeled probe for detection of rt204 mutation in HBV P gene.Methods Plasmids with wild strain rt204M,mutant strains rt204I and rt204V were constructed,and the probes were designed and optimized.HRM plots were established by the constructed plasmids.A total of 185 samples were collected from patients with chronic hepatitis B in the Affiliated Hospital of School of Medicine of Ningbo University during May 2010 and May 2012.All samples were detected by HRM,and matched with characteristic melting pattern.rt204 mutations were screened,and then verified by DNA sequencing.Paired x2 test was used for the comparison of the detection of mutations.Results The melting temperatures for rt204I,rt204V and rt204M were 58.0℃,60.6℃ and 62.5℃,respectively.Among 185 samples,168 samples (90.8％) could be analyzed by HRM method,and 155 samples (83.8％) coule be successfully sequenced (P ＜0.01).In the 155 samples which were completely analyzed by HRM assay and sequencing,75 samples were rt204M,55samples were rt204I,and 25 samples were rt204V by using HRM method,with an overall mutation detection rate of 51.6％ (80/155) ; and by sequencing,110 samples were rt204M,30 samples were rt204I,and 15samples were rt204V,with an overall mutation detection rate of 29.0％ (45/155).The difference onmutation rates detected by the above two methods was of statistical significance (P ＜ 0.01).Conclusion HRM with unlabeled probe is simple,sensitive,rapid and specific for detection of rt204 mutation in HBV P gene.