通过增加痰液量和超声裂菌提高结核分枝杆菌荧光定量PCR检出率

摘要

Objective To increase the detection rate of Mycobacterium tuberculosis(MTB)in real-time fluorescence quantitation polymerase chain reaction (PCR)by increasing sputum volume and modifying ultrasonic extraction of MTB DNA.Methods Sputum samples from 206 tuberculosis patients and 103 non-tuberculosis patients as controls were analyzed.Routine sputum MTB smear and liquid culture of MTB by American BD MAGIT960 system were performed.At least 5mL sputum was collected from each patient,and 2-3-fold volume of 4% NaOH solution was added to the sputum. After liquefaction,1 mL solution was used for the routine DNA extraction procedure,and the remaining part was used for the modified ultrasonic extraction procedure.Both of the extracted DNA were quantitated by real-time fluorescence quantitation PCR.Results By the modified ultrasonic extraction procedure,the MTB DNA positive detection rate increased from 87.5%(confidence interval 81.4%-93.6%)to 95.5%(confidence interval 91.7%-99.4%)(P<0.05)in the smear positive and culture positive tuberculosis patients,and the positive detection rate increased from 57.4%(confidence interval 47.5%-67.4%)to 83%(confidence interval 75.4%-90.6%)in the smear negative and culture positive tuberculosis patients (P<0.01 ).The quantity results of increasing sputum volume and modifying ultrasonic extraction procedure increased 14 folds in average by the modified ultrasonic extraction procedure.Conclusions The increasing sputum volume and modifying ultrasonic extraction procedure increase the positive detection rate of real-time fluorescence quantitation PCR for tuberculosis,which can be recommended for routine laboratory use.%目的：通过增加痰液量和超声破碎法提取痰液中的结核菌DNA，提高结核分枝杆菌实时荧光定量聚合酶链反应（PCR）的检出率。方法选择206例肺结核患者，以103例非结核患者作为对照组；肺结核患者同时进行结核菌涂片，并在美国BD公司的MAGIT960仪器上进行液体快速培养结核菌。另收集至少5 mL的痰液，加入2～3倍体积的4％氢氧化钠液化后，取1 mL按照采用常规方法抽提痰液中的结核菌DNA，剩余部分采用改进的超声破碎法提取结核菌DNA；两者同时进行实时荧光定量PCR检测。结果采用增量超声法，对于涂片阳性、培养阳性的结核患者，阳性检出率由87．5％（可信区间为81．4％～93．6％）提升至95．5％（可信区间为91．7％～99．4％）（P＜0．05）。对于涂片阴性、培养阳性的患者，阳性检出率由57．4％（可信区间为47．5％～67．4％）提高至83％（可信区间为75．4％～90．6％）（P＜0．01）。增量超声法比常规方法，定量值平均提高14倍以上。结论通过增加痰液量和超声破碎法提取痰液中的结核菌DNA，可提高实时荧光定量PCR结核分枝杆菌的检出率，值得进一步推广应用。