| Objective:Genotypic resistance mutation is the major cause of hepatitis B virus (HBV) drugresistance. Although DNA sequencing has been widely used for the discovery ofresistance mutations, its clinical application is largely limited by the inherent lowsensitivity. Reverse hybrid technique has higher sensitivity (about5%), but itsapplication is hampered by the high cost and complicated operation system, making itdifficult to be used widely in developing countries in particular. Other newtechnologies such as mass spectrometry, ultra-deep sequencing and high throughputsequencing have gradually come into use. However, the high cost, complexity inoperation and massive data interpretation make them preferred more for researchpurposes, rather than large clinical applications. In order to overcome the limitationsof the methods stated above, this study has established a highly sensitive COLD-PCR(coamplification at lower denaturation temperature PCR) method in combination withSanger sequencing technology, aiming to detect known and unknown HBV genotypicresistant mutations.Methods:1.Primers were designed based on the common mutations of HBV reversetranscriptase (rt) which can simultaneously amplify both wild and mutant HBV DNArt180~rt215. HBV mutants were amplified by modification of PCR conditions(mostly denaturation temperature) to determine critical denaturation temperature (Tc).2. Tc was established as a denaturing temperature for both Fast and FullCOLD-PCR. The Fast COLD-PCR and Full COLD-PCR (hybridization added to enrich all mutations) in conjunction with Sanger sequencing were compared withconventional PCR/Sanger sequencing, in terms of the sensitivity, the specificity andthe minimum detection limit for mutants.3. The mutant frequency for HBV genotypic resistance was analyzed amongConventional PCR, Fast COLD-PCR and Full COLD-PCR in combination withSanger sequencing in30cases of chronic hepatitis B (CHB) with virologicaloutbreak.Results:1. The sensitivity of the conventional PCR/Sanger for all types of mutant was10%(mutant:wild type=1:10, the same for below). For single base pair mutation,when the Tm-reducing mutation occured (such as C:G→T:A), the sensitivity of Fastand Full COLD-PCR/Sanger for mutant detection were1%and2%, respectively;when the mutant caused no change of Tm (such as C:G→G:C) or raising Tm (T:A→C:G), only Full COLD-PCR/Sanger improved the mutant detection sensitivity(2%). For combination mutation, the sensitivity of Fast COLD-PCR/Sanger and FullCOLD-PCR/Sanger can be raised to0.5%and1%, respectively.2. The concentration required for Fast COLD-PCR/Sanger varies for differenttypes of mutations, the minimum detection limit of the mutant DNA ranged from5.0E+01IU/ml to1.0E+03IU/ml. The concentration required for FullCOLD-PCR/Sanger was approximately the same for single mutation, and wasdetectable with the mutant DNA concentrations≥2.0E+02IU/ml; the minimumdetection limit for combination mutation was lowered to1.0E+02IU/ml.3. In30CHB patients, no HBV gene mutations were detected by conventionalPCR/Sanger sequencing. Whereas21mutations were successfully detected byCOLD-PCR/Sanger sequencing. In which Fast COLD-PCR/Sanger sequencingdetected mutations in17cases (5cases of rtA181T+rtM204I,3cases of rtA181T,3cases of rtM204I,2cases of rtV191I,2cases of rtH197H,1case of rtQ182Q+rtV191I and1case of rtM204I+rtK212K); Full COLD-PCR/Sanger sequencingdetected mutations in14cases (4cases of rtM204I,2cases of rtV191I,1case of rtA181T,1case of rtH197H,2cases of rtS189S+rtM204I,1case rtA181T+rtM204I,1case rtA180T+rtM204V,1case rtQ182Q+rtV191I and1casertS213S).Conclusions:The major improvement in sensitivity provided by COLD-PCR, defined by Fastand Full mode has enabled the common Sanger sequencing method to be morepowerful in detecting low-level mutations in clinical CHB samples. COLD-PCRshowed unbeatable advantages of being simple, practical and inexpensive, which isparticularly ideal for application in clinical laboratories. |
