应用于结核性胸膜炎分子生物学检测的胸腔积液核酸提取方法初步比较分析北大核心CSCD

目的筛选鉴定有助于结核性胸膜炎分子生物学检测的胸腔积液核酸提取方法。方法收集18例结核性胸膜炎患者胸腔积液样本,分别采用6种方法提取核酸,应用微滴数字PCR技术分析样本中结核分枝杆菌特异性核酸IS6110和IS1081数量,以此评估不同胸腔积液核酸提取方法的优劣。结果对于胸腔积液原液或胸腔积液离心上清,采用柱法和磁珠法提取的核酸样本中靶标数量无明显差别。对于胸腔积液离心沉渣,采用物理破碎法提取的核酸样本中IS6110和IS1081数量显著高于化学破碎法(IS6110,Z=-3.408,P=0.001;IS1081,Z=-3.297,P=0.001)。相比于小体积(0.7 mL)胸腔积液原液直接提取,大体积(4 mL)胸腔积液离心的上清和沉渣(物理破碎)中提取的核酸样本含有更多的靶标数量(分别IS6110,Z=-3.237,P=0.001,IS1081,Z=-2.667,P=0.008;IS6110,Z=-3.157,P=0.002,IS1081,Z=-2.250,P=0.024)。结论大体积胸腔积液离心上清游离核酸提取法和离心沉渣物理破碎核酸提取法能够富集更多的结核分枝杆菌核酸,是较好的用于结核性胸膜炎分子生物学检测的核酸制备方法。

Comparative analysis of methods for nucleic acid extraction from pleural effusion applied to molecular biological detection of tuberculous pleurisy

To identify a pleural effusion nucleic acid extraction method to improve the molecular biological detection of tuberculous pleurisy, we collected 18 pleural effusion samples from patients with tuberculous pleurisy and used six methods to extract nucleic acids from each sample. The copy numbers of the Mycobacterium tuberculosis specific nucleic acids IS6110 and IS1081 in samples were quantified with droplet digital polymerase chain reaction (PCR). The nucleic acid extraction methods were evaluated by comparison of the number of detected targets. For pleural effusion or pleural effusion supernatants, no significant difference was observed between the column method and magnetic bead method. For pleural effusion precipitates, the copy number of IS6110 and IS1081 was significantly higher in nucleic acid samples extracted with the physical disruption method than the chemical lysis method (IS6110, Z=-3.408, P=0.001;IS1081, Z=-3.297, P=0.001). Compared with nucleic acid samples directly extracted from small volume (0.7 mL) pleural effusion, more targets were detected in nucleic acid samples extracted from large volume (4 mL) pleural effusion, in both supernatants and precipitates (physical disruption) (IS6110, Z=-3.237, P=0.001, IS1081, Z=-2.667, P=0.008; IS6110, Z=-3.157, P= 0.002, IS1081, Z=-2.250, P=0.024, respectively). In conclusion, the supernatant circulating nucleic acid extraction method and precipitate physical disruption nucleic acid extraction method from massive pleural effusion enriched more M. tuberculosis nucleic acids, thus providing a better method for molecular biological detection of tuberculous pleurisy.