一种基于Propidium Monoazide与PCR结合的选择性检测细菌活细胞的方法

建立了一种PMA与PCR结合的细菌活细胞检测的方法，可有效抑制微生物死细胞DNA的PCR扩增，从而排除死细胞对微生物活细胞检测的影响．结果表明，PMA浓度大于3 μg/ml时可抑制死细胞DNA的PCR扩增，PMA浓度高达50 μg/ml时仍不影响活细胞DNA的PCR扩增；并且得出曝光时间大于3 min可使PMA与死细胞DNA分子共价交联，抑制其PCR扩增；浊度影响PMA交联的结果表明浊度小于10 NTU时，PMA仍能有效地抑制死细胞DNA的PCR扩增，而当浊度大于100 NTU时，PMA失去效果．

Method for bacterial viable cell selective detection basing on Propidium Monoazide in combination with PCR

Propidium Monoazide (PMA) is a kind of DNA-intercalating dyes that penetrate only into dead cells with compromised cell membrane integrity but not into viable cells with intact cell membranes. After intercalating in the DNA of dead cells, the photoinducible azide group is inverted to highly reactive nitrene radical by exposure to bright light, and readily reacts with hydrocarbon moiety at the binding site to form a stable covalent nitrogen-carbon bond, which inhibiting the PCR of DNA molecular. A method that PMA combine with PCR is useful for selective detecting microbial viable cell from viable cells and dead cells mixture. This study demonstrated that the concentration of PMA greater than 3μg/ml could inhibit the PCR amplification of DNA from dead cells. PMA concentration at 50μg/ml or less than 50μg/ml had little or no inhibition on the PCR amplification of DNA from viable cells. Light exposure time more than 3 min could inhibit the PCR amplification of DNA from dead cells. PMA treatment was effective when turbidities less than 10 NTU, PMA treatment have no effect when turbidities larger than 100 NTU.