Comparative analysis of methods for nucleic acid extraction from pleural effusion applied to molecular biological detection of tuberculous pleurisy
DU Bo-ping, LI Zi-hui, PAN Li-ping, SUN Qi, LYU Ling-na, ZHU Chuan-zhi, XING Ai-ying, JIA Hong-yan, ZHANG Zong-de
Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing 101149, China
Abstract: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.
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