Establish Allele Specific Locked Nucleic Acid Real Time Quantitative PCR For Detection Of HBV Adefovir Dipivoxil Resistance Mutations And Evaluate Its Clinical Application

ObjectivesEstablish allele specific locked nucleic acid real time quantitative PCR(RT-AS-LNA-q PCR) for detection of HBV Adefovir Dipivoxil resistance mutations and evaluate its clinical application.Methods1 Establish RT-AS-LNA-q PCR for detection of HBV Adefovir Dipivoxil resistance mutation1.1 Establishment of RT-AS-LNA-q PCR methodologyIncluding reaction system and amplification condition, the preparation of standard plasmids and standard curve, etc.1.2 Evaluation of RT-AS-LNA-q PCRReorganization of the wild type and mutant plasmid standard was tested by RT-AS-LNA-q PCR(1×1010copies/?l~1×101 copies/?l) and evaluate its linear range, specificity, sensitivity, repeatability, accuracy and sensitivity of detection of HBV DNA mutation type.1.3 Methodological comparisonClinical specimens were parallel detected by Sanger sequencing and self-built method, Cohen's Kappa test was performed to test their performance.Clinical samples containing low level HBV DNA mutations detected by self-built method and cloning sequencing to test the accuracy of the self-built method.1.4 Clinical evaluation of RT-AS-LNA-q PCRUsing self-built method tested clinical mixed templates containing different proportion of mutations to determine the precise and stable in detecting the minimum mutation rate, and evaluate its sensitivity used in the detection of clinical samples;Results1 To establish RT-AS-LNA-q PCR detection of HBV ADV mutation1.1 Established RT-AS-LNA-q PCR successfullyThe standard plasmid and standard curve of HBV ADV rt A181 V and rt N236 T mutation was prepared successfully.The reaction system and amplification condition is determined.1.2 Evaluation of RT-AS-LNA-q PCR methodRT-AS-LNA-q PCR detected ADV 181 loci and 236 loci of wild type and mutant standard plasmid whose linear range was between 1×109copies/?l and 1×102 copies/?l. The intra-run and inter-run coefficient of variation(CV) of 181 loci was between 1.61% and 12.8%. The intra-run and inter-run coefficient of variation(CV) of 236 loci was between 3.22% and 13.85%. Sensitivity of the 181 and 236 loci were 10-5 in the background of 1×107 copies/?l wild-type standard plasmid.1.3 Methodological comparisonRT-AS-LNA-qPCR and Sanger sequencing assay mutation of 181 loci, fully consistent results accounted for 88.2%(90/102), partial consistent results accounted for 11.8%(12/102), without completely inconsistent results(0/102), the two methods showed good consistency(P= 0.000).RT-AS-LNA-q PCR and Sanger sequencing assay mutation of 236 loci, fully consistent results accounted for 91.2%(93/102), partial consistent results accounted for 8.8%(9/102), without completely inconsistent results(0/102). the two methods showed good consistency(P= 0.000).RT-AS-LNA-q PCR results were completely consistent with the cloning sequencing.1.4 Clinical evaluation of RT-AS-LNA-q PCRThe sensitivity of the assay in detection of clinical samples was 0.04%(181 loci)?0.05%(236 loci). RT-AS-LNA-q PCR was a high sensitive assay which could applied to early detect HBV drug-resistance mutants in clinic.Self-built method used to detect the clinical specimens, the sensitivity was 0.04%(181 loci), 0.05%(236 loci). The sensitivity of RT-AS-LNA-q PCR is high which can be used in detecting early drug resistance mutation of ADV.ConclusionsA novel assay named RT-AS-LNA-q PCR for detection of HBV rt A181 V and rt N236 T mutation was successfully established. The assay had advantages over direct sequencing which could applied widely in clinical laboratories with wide linear range, specific, high sensitive, reproducibility and reliable, etc.Successfully established RT-AS-LNA-q PCR method for detecting HBV rt A181 V and rt N236 T resistance mutations. The method had wide linear range, good specificity, high sensitivity, good reproducibility, which was better than Sanger sequencing and suitable for general genetic diagnostic laboratory.