乙型肝炎病毒耐药突变检测方法的创新与标准化研究

HBV genotypic resistance resistance develops earlier than virological and biochemical breakthrough during the theatment of chrnic hepatitis B with nucleos(t)ide analogs (NAs) and helps confirm the drug resistance at the very early stage and guide the rescue therapy. But the methods of detecting genotypic resistance used nowadays are neither sensitive nor specific enough, which limits their usefulness. Our preliminary studies have shown that the sensitivity of direct sequencing is low and the real-time PCR, DNA microarray and line probe assay(LiPA) often yield false negative results or indeterminate ones due to the DNA polymorphisms of HBV strains in Chinese patients. So it is urgent to optimize and standardize these methods. We aim to use COLD-PCR (coamplification at lower denaturation temperature PCR) in combination with pyrosequencing in place of direct sequencing to improve the sensitivity in detecting minor mutation. Meanwhile we will find the HBV DNA polymorphisms that affcet the performance of resistance detecting methods such as real-time PCR, microarray and LiPA.Subsequently new probes will be introduced to improve their detection efficiency. After the methodological optimization, we will use WHO standard and the serum panals for HBV to set up the performance parameters of different methods in order to standardize the resistance detection methods. Based on the standardized methods, we will set up the cut-off values for HBV genotypic resistance and quantify the levels of resistance quasispecies to investigate the development and its clinical significance of minor mutions confering drug resistance during the antiviral therapy with NAs.

慢性乙型肝炎核苷（酸）类似物抗病毒治疗过程中，基因型耐药先于病毒学和生化学突破出现，更有利于早期指示耐药产生。但现有检测方法的性能如灵敏度及特异性等存在局限，制约其临床应用；耐药与突破的关系也有待进一步探究。我们前期研究表明，传统PCR测序检测微量耐药突变的灵敏度较低，而实时荧光PCR、基因芯片及线性探针法易受中国人群特异性HBV 多态性位点影响出现假阳性或不确定结果。我们采用低变性温度共扩增PCR（COLD-PCR）联合焦磷酸测序法建立新型高灵敏度耐药检测方法，同时探寻影响检测特异性的多态性位点并设计新型辅助型探针和引物以提高检测特异性。之后应用HBV 国际标准品和耐药血清盘建立各方法的检测性能参数体系，推动方法学标准化。在此基础上建立判定耐药的相对和绝对定量cut-off值，进一步规范其临床应用，并研究微量突变准种的产生与动态变化规律，为阐明病毒学突破的机制、探寻潜在突变位点提供新思路

背景：现有各指南均还未推荐将基因型耐药检测作为核苷(酸)类似物抗病毒治疗常规检测，主要原因是现有的HBV 基因型耐药检测方法的操作与结果的判定均缺乏标准化，检测性能（灵敏度、特异性等）存在种种不足，且各方法间缺乏直接比较，检测方法的性能参数并未建立，因此阻碍了其临床广泛应用，.主要研究内容及重要结果：首先，通过本项目的资助，成功构建课题相关的临床样本库和数据库。其次，完成了HBV耐药检测的方法学研发，内容包括：①构建COLD-PCR技术，成功实现A181+M204位点和N236+M250位点的扩增，实现了P区大片段的共扩增。②研发了新型的基于pyrosequencing技术HBV耐药检测试剂盒，能较为准确反映HBV耐药毒株所占的比例；③建立在我国临床实验室广泛使用的HBV耐药检测方法的性能参数体系，完善了影响HBV耐药检测方法灵敏度或特异性的中国人群HBV DNA序列特征。④研究了HBV突变对临床检验常规检测项目的影响，结果表明常见HBV突变对HBsAg定量等检测试剂并没有影响。⑤探究新的强效抗HBV药物耐药位点，已纳入124例慢性乙型肝炎患者，对患者的病毒载量、基因型和耐药位点进行定期监测。目前为止尚未发现出现病毒学突破和潜在耐药的患者。⑥建立了以Illumina公司最新的NextSeq 500二代测序仪为基础的二代测序平台，并接受卫生计生委临床检验中心每年定期的质控评审，目前为止，所有质控项目均为“合格”。⑦对HBV耐药突变检测新技术的进行肝病研究相关的临床应用。.关键数据及其科学意义：HBV耐药检测的灵敏度目前可以达到0.1%-1%左右，相比传统的HBV耐药检测技术，标准化、高灵敏度的HBV耐药检测平台可以更早地预测HBV病毒学突破，及时转换抗病毒治疗的方案，造福广大慢性乙型肝炎患者。