慢性乙型肝炎（CHB）患者核苷（酸）类似物（NAs）治疗过程中，HBV逆转录酶（RT）区准种检测对于预测NAs疗效有重要价值。然而，由于不同基因型HBV准种存在复杂性和多样性，目前尚无法常规高通量定量检测准种。前期我们已成功构建低变性温度共扩增PCR（COLD-PCR）用于HBV DNA多位点突变的定性检测。本研究拟采用可富集低比例突变DNA的COLD-PCR法，结合型特异性探针熔解曲线，建立一种多位点突变联合定量检测的COLD-PCR/探针熔解曲线技术，实现一次反应同时检测HBV基因型、突变类型及突变DNA的比例，以评估HBV RT区的准种特点。该技术具有高灵敏度、高通量、简便实用和价廉的优点。应用该体系对CHB患者HBV RT区准种进行动态研究，有助于探清准种发生发展的过程、特点和规律，揭示其变化与NAs疗效之间的关系，为不同HBV基因型感染的CHB患者实施个体化诊疗奠定基础。

Detection of hepatitis B virus (HBV) quasispecies (QS) in the reverse transcriptase (RT) region has significant predicted values during nucleos(t)ide analogues (NAs) treatment in chronic hepatitis B patients. However, until now, there is no high-throughput technology for the detection of QS in the clinic due to the complexity and diversity of HBV QS. In our previous work, we have successfully established COLD-PCR for qualitative detection of multiple mutations. In this study, we intend to establish COLD-PCR in conjunction with probe-based fluorescence melting curve analysis (FMCA) technology to evaluate the characteristics of HBV QS. We develop a highly sensitive COLD-PCR to improve the amplification efficacy in detecting low-level mutations and combine with type-specific FMCA to analyze HBV genotype, variety and proportion of mutations to establish a highly sensitive, simple, inexpensive, practical and high-throughput tool for quantitative detection of multiple mutations in clinic. Dynamic study of the HBV RT QS by this novel method can help to explore the characteristics and regularities of the development of HBV QS, also to discover the relationship between HBV QS and the curative effect with NAs, which may lay a foundation for personalized diagnoses and treatment in the CHB patients.