【摘要】 目的探讨经煮沸、Chelex100树脂处理、核酸纯化三种方法平行处理的标本在进行结核分枝杆菌蝎型探针荧光定量聚合酶链反应(PCR)检测中的有效性,以得到标本的最佳处理效果。方法收集42例结核确诊患者的多种标本和14例非肺结核患者的痰标本,对其分别用上述3种方法平行处理后,采用蝎型探针荧光定量PCR法进行检测,并分析其结果。结果核酸纯化法处理的标本阳性检出率为100%,明显高于煮沸法57.14%和Chelex100树脂法52.38%(分别为P=0.0103,P=0.0233);尤其是外观呈血性和菌阴性的标本,采用核酸纯化法处理标本后检测所得结核DNA量较其他方法处理所得量高约1~2个数量级。结论标本的处理对于结核分枝杆菌蝎型探针荧光定量PCR检测十分重要,煮沸(裂解)法和Chelex100树脂法不适合用以处理临床标本,而应使用核酸纯化法。更多还原

【Abstract】 Objective To compare the effects of extracting bacteria DNA from clinical samples processed by boiling,Chelex 100 resin treatment and bacterial DNA Mini kit respectively in detecting Mycobacterium tuberculosis by fluorescence quantitative PCR(FQ-PCR) using Duplex Scorpion Primer.Methods The clinical samples of 42 patients with tuberculosis and 14 patients with non-tuberculosis were processed by above three methods respectively,and FQ-PCR was performed on the ABI7000 using primers targeting an 55-bp fragment of TB senX3-regX3 IR.Results The positive detection rate of Mycobacterium tuberculosis from samples processed by bacterial DNA Mini kit was 100%,which was significantly higher than that of boiling(57.14%) and Chelex 100 resin(52.38%) treatment methods(P=0.0103,P=0.0233,respectively).Conclusion The effectiveness of clinical sample preparation is important for FQ-PCR using Duplex Scorpion Primer.The DNA templates prepared by boiling and Chelex 100 resin treatment are not suitable for Mycobacterium tuberculosis DNA quantitative PCR detection,and nucleic acids for PCR must be purified from clinical samples,which is necessary for the accuracy of the FQ-PCR assay.更多还原