反向杂交检测乙型肝炎病毒耐药突变方法的优化及其初步评估

目的 建立一种HBV耐药突变的反向杂交检测方法,并对该方法进行优化和评估.方法 针对拉米夫定、阿德福韦酯和恩替卡韦3种药物10个耐药位点的常见耐药突变形式,合成兼顾HBV 8种基因型的26条简并探针,并固定于同一张带正电荷的尼龙膜上,与地高辛标记的待测样本聚合酶链反应(PCR)产物进行杂交.为了提高检测灵敏度和特异性,从PCR产物标记地高辛的数目,紫外交联探针的能量强度,以及杂交和严格洗脱4个方面进行优化.为了证实方法的可行性,从检测特异性、灵敏度和临床标本的检测准确性3个方面进行评估.结果 当检测体系为引物标记3个地高辛分子,紫外交联的能量强度选择1500× 0.1 mJ/cm2,杂交温度选择42℃,严格洗脱条件选择44℃,用0.5 ×SSC和0.1％十二烷基硫酸钠严格洗脱30 min时,可获得灵敏、特异的结果.在该检测体系的评估中,当PCR产量在10 mg/L以上,可以被检测到,且绝大多数探针特异性较好.临床标本的检测结果与直接测序法的检测结果符合率为93.9％ (31/33).结论 该方法可以对HBV拉米夫定、阿德福韦和恩替卡韦耐药突变同时进行检测,是一种简便、快速、灵敏的分析方法,但部分探针的特异性有待进一步提高.对于180/181、202/204这4个位点的探针,由于两两距离较近,同一探针序列包含2个位点的密码子,杂交会相互干扰,通过组合距离较近的2个位点的密码子的各种形式来设计探针,可能有助于取得更好的结果.

Optimization and assessment of a reverse hybridization system for the detection of HBV drug-resistant mutations

Objective To establish a detection method for HBV drug-resistant mutations related to lamivudine,adefovir and entecavir by optimization and assessment of reverse hybridization system.Methods 26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane.PCR products labeled with digoxigenins were hybridized with corresponding probes.To improve the sensitivity and specificity,4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin,the energy intensity of UV cross-linking,hybridization and stringency wash conditions.To prove the feasibility,the specificity,semitivity and accuracy of this system were assessed respectively.Results Sensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenins,energy intensity of UV cross-linking for 1 500 × 0.1 mJ/cm2,hybridization at 42℃ and stringency wash with 0.5 × SSC and 0.1％ SDS solution at 44℃ for 30min.In the assessment of system,the majority of probes have high specificity.The quantity of PCR product with a concentration of 10 ng/μl or above can be detected by this method.The concordant rate between reverse hybridization and direct sequencing is 93.9％ in the clinical sample test.Conclusion Though the specificity of several probes needs to be improved further,it is a simple,rapid and sensitive method which can detect HBV resistant mutations related to lamivudine,adefovir and entecavir simultaneously.Due to the short distance between 180 and 181,likewise 202 and 204,the sequence of the same probe covers two codon positions,and hybridization will be interfered by each other.To avoid such interference,the possible solution is that probes are designed by arranging and combining various forms of two near codons.