Design and validation of a clinical DNA sequencing assay for drug-resistance genes in hepatitis B virus

Objective Under the ISO15189 and America Association of Pathologists ( CAP ) laboratory accreditation system, to establish the performance verification standards for detecting hepatitis B virus resistance gene by sanger sequencing.Methods 25 cases of HBV drug resistance outpatients and inpatients were collected from August 2012 to December in Hepatitis Clinic of Huashan Hospital Affiliated to Fudan University.Analytical performance parameters including analytical sensitivity, precision, accuracy, analytical specificity, reference range/reportable range, etc were evaluated.Sequencing quality evaluation parameters included fluorescence signal intensity overall sequencing chromatogram, signal to noise ratio, trace scores and QV value.Results 10%-20% mutation could be detected under wild-type background. The methool had good precision and accuracy.No obvious interference and cross contamination were observed.Conclusions Performance validation of the sequencing should combine with the practical application.Especially in view of the different detection subjects, and appropriately adjusted to meet the clinical needs.Detection of hepatitis B virus resistance gene by the in the test method in this study can be used in clinical detection.