Taqman MGB探针荧光定量PCR检测HBVDNA含量及YMDD变异的方法学评价

Evaluation of Taqman MGB probe fluorescence quantitative PCR detection of HBV DNA content and YMDD mutation

目的评价Taqman MGB探针荧光定量PCR技术检测血清乙型肝炎病毒DNA含量及YMDD变异的方法学特性。方法照美国临床和实验室标准协会（clinical and laboratory standards institute,CLSI）制定的评价方案,对该方法HBV DNA定量的线性范围、精密度、准确性作评价。以序列直接测定法作为诊断YMDD变异的金标准,对该方法YMDD变异检测的灵敏度、特异性和诊断符合率作评价。结果该方法HBV DNA定量在10^3-10^8copy/ml范围内呈线性,精密度好（〈10%）,准确度高（平均回收率为97.5%）。CT（FAM）≤35,Ct设定临界值为7时,当HBV YMDD变异〉25%时能检测到变异存在;Ct设定临界值为UNDETECT时,当HBV YMDD变异〉50%时能检测到变异存在。以直接测序方法为金标准,该方法YMDD变异检测的灵敏度97%,特异性100%,诊断符合率99.5%。结论该方法检测HBV DNA载量和YMDD变异可以在同一管中完成,特异性高,灵敏度相对较低,但具有快速、准确、经济实用等特点。

Objective To evaluate methodology of the Taqman MGB probe fluorescence quantitative PCR detection of serum HBV DNA content YMDD mutation . Methods Use clinical and laboratory standards institute （CLSI） evaluation programme to evaluate the HBV DNA quantitative methods linear range, precision and accuracy.Directsequence method to YMDD mutation as a diagnostic gold standard, to evaluate this methods sensitivity, specificity and diagnostic accuracy . Results This quantitative method of HBV DNA was linear in the range 10^ 3 - 10^ 8 copy / ml, good precision （CV 〈10%）, high accuracy （average recovery of 97.5%）. CT （FAM） ≤ 35, when Ct threshold set for 7.0,HBV YMDD mutation〉 25% can be detected; when Ct set thresholds for UNDE- TECT, HBV YMDD mutation〉 50% can be detected variation. Direct sequencing approach to the standard,this method of YMDD mutation detection sensitivity 97%, specificity 100%, the diagnosis rate of 99.5%. Conclusion This detection of HBV DNA and YMDD mutation contained in the same tube can be completed. This detection of YMDD mutationsin high specificity and sensitivity relatively low, but is fast, accurate, economical and practical characteristics and suitable for clinical laboratories, worthy of promotion.