CAJ | 학술논문

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。
목적：통과증가담액량화초성파쇄법제취담액중적결핵균DNA，제고결핵분지간균실시형광정량취합매련반응（PCR）적검출솔。방법선택206례폐결핵환자，이103례비결핵환자작위대조조；폐결핵환자동시진행결핵균도편，병재미국BD공사적MAGIT960의기상진행액체쾌속배양결핵균。령수집지소5 mL적담액，가입2～3배체적적4％경양화납액화후，취1 mL안조채용상규방법추제담액중적결핵균DNA，잉여부분채용개진적초성파쇄법제취결핵균DNA；량자동시진행실시형광정량PCR검측。결과채용증량초성법，대우도편양성、배양양성적결핵환자，양성검출솔유87．5％（가신구간위81．4％～93．6％）제승지95．5％（가신구간위91．7％～99．4％）（P＜0．05）。대우도편음성、배양양성적환자，양성검출솔유57．4％（가신구간위47．5％～67．4％）제고지83％（가신구간위75．4％～90．6％）（P＜0．01）。증량초성법비상규방법，정량치평균제고14배이상。결론통과증가담액량화초성파쇄법제취담액중적결핵균DNA，가제고실시형광정량PCR결핵분지간균적검출솔，치득진일보추엄응용。
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.