摘要目的:建立基于微流控芯片式数字PCR（cdPCR）的乙型肝炎病毒（HBV）RNA检测方法，并评估其在慢性HBV感染者中的应用。方法:选取福建医科大学附属第一医院的135例慢性HBV感染者，根据HBV DNA含量分为HBV DNA>100 IU/ml组（85例）和HBV DNA≤100 IU/ml组（50例），同时选取健康体检者及其他病毒感染者作为对照组（15例）。设计HBV前C/C区特异性引物和探针，优化微流控cdPCR检测方法，并评估其线性范围、检出限、特异性及精密度。使用自建方法和2种市售试剂盒分别检测患者血清HBV RNA含量。采用Pearson相关性分析对HBV RNA与其他HBV标志物的关系进行评估。结果:优化后自建微流控cdPCR方法的变性时间为10 s，退火/延伸温度为62 ℃，引物和探针浓度分别为0.3和0.2 μmol/L。其线性范围为103~10 7拷贝/ml，检出限为102拷贝/ml，10 4和10 7拷贝/ml质粒标准品的批内变异系数（ CV）分别为1.06%和0.82%，批间 CV分别为0.75%和0.44%。特异性实验显示，其他病原体感染者和健康体检者的血清中均未检出阳性信号。自建微流控cdPCR方法在HBV DNA>100 IU/ml组的检出率为81.18%（69/85），高于市售试剂盒B的64.71%（55/85）（ P<0.016 7）。在HBV DNA≤100 IU/ml组中，3种方法的检出率差异无统计学意义（ P>0.05）。HBV RNA与HBV DNA、乙型肝炎表面抗原及乙型肝炎e抗原均呈正相关（ r=0.67、0.53、0.44， P均 <0.001）。 结论:成功建立了微流控cdPCR定量检测HBV RNA的方法，该方法灵敏、特异，且在低浓度病毒载量样本中也具有良好的检测能力。

abstractsObjective:To establish a microfluidic chip-based digital PCR (cdPCR) method for detecting hepatitis B virus (HBV) RNA and evaluate its application in patients with chronic HBV infection.Methods:A total of 135 patients with chronic HBV infection were recruited from the First Affiliated Hospital of Fujian Medical University and stratified into two groups based on HBV DNA levels: HBV DNA>100 IU/ml ( n=85) and HBV DNA≤100 IU/ml ( n=50). Additionally, healthy individuals and subjects infected with other viruses ( n=15) served as controls. Primers and probes targeting the HBV pre-C/C region were designed to optimize the microfluidic cdPCR method, and its linear range, limit of detection (LOD), specificity, and precision were assessed. Serum HBV RNA levels were measured using the self-developed method and two commercial kits. Pearson correlation was applied to evaluate the relationships between HBV RNA and other HBV markers. Results:The optimized microfluidic cdPCR method featured a denaturation time of 10 seconds, an annealing/extension temperature of 62 ℃, and primer and probe concentrations of 0.3 μmol/L and 0.2 μmol/L, respectively. The method demonstrated a linear range of 103-10? copies/ml and an LOD of 102 copies/ml. The intra-assay coefficient of variation ( CV) for plasmid standards at 10? and 10? copies/ml were 1.06% and 0.82%, respectively, while the inter-assay CVs were 0.75% and 0.44%. Specificity analysis confirmed the absence of positive signals in sera from healthy controls and subjects infected with other pathogens. In the HBV DNA>100 IU/ml group, the detection rate of the self-developed cdPCR method was 81.18% (69/85), significantly higher than the 64.71% (55/85) achieved by commercial kit B ( P<0.016 7). However, in the HBV DNA≤100 IU/ml group, no significant differences were observed among the three methods ( P>0.05). HBV RNA levels were positively correlated with HBV DNA ( r=0.67), hepatitis B surface antigen ( r=0.53), and hepatitis B e antigen ( r=0.44) (all P<0.001). Conclusion:A microfluidic cdPCR assay for the quantitative detection of HBV RNA has been successfully developed. This assay demonstrates high sensitivity, specificity, and robust detection capability, even for low HBV DNA-concentration samples.