College of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, People's Republic of China.
Appl Microbiol Biotechnol. 2010 Mar;86(1):377-84. doi: 10.1007/s00253-009-2373-1. Epub 2009 Dec 19.
Propidium monoazide can limit the analysis of microbial communities derived from genetic fingerprints to viable cells with intact cell membranes. However, PMA treatment cannot completely suppress polymerase chain reaction (PCR) amplification when the targeted gene is too short. PMA treatment in combination with two-step nested PCR was designed to overcome this problem. Four experiments were performed to determine the limitation of PMA treatment and to evaluate the suitability of the method by applying the following samples: (1) pure cultures of Escherichia coli O157:H7, Enterobacter aerogenes, and Alcaligenes faecalis; (2) pond water samples spiked with heat-killed E. coli O157:H7 and E. aerogenes; (3) anaerobic sludge samples exposed to increasing heat stress; and (4) selected natural samples of estuarine sediment and lake mud. Results from the first two experiments show that PMA treatment cannot efficiently suppress dead cells from PCR amplification when the targeted gene is as short as 190 bp, however, the two-step nested PCR can overcome this problem. The last two experiments indicate the method that PMA treatment in combination with two-step nested PCR is useful for viable cells detection in microbial ecology.
吖啶橙单染色法可以将遗传指纹分析的微生物群落限制在具有完整细胞膜的存活细胞上。然而，当目标基因过短时，吖啶橙处理不能完全抑制聚合酶链反应（PCR）扩增。设计了吖啶橙处理与两步嵌套 PCR 相结合的方法来克服这个问题。进行了四项实验来确定吖啶橙处理的限制，并通过以下样本评估该方法的适用性：（1）大肠杆菌 O157:H7、产气肠杆菌和粪产碱杆菌的纯培养物；（2）用热灭活的大肠杆菌 O157:H7 和产气肠杆菌接种的池塘水样本；（3）暴露于逐渐增加的热应激的厌氧污泥样本；和（4）选定的河口沉积物和湖泥自然样本。前两个实验的结果表明，当目标基因短至 190bp 时，吖啶橙处理不能有效地抑制死细胞的 PCR 扩增，但是两步嵌套 PCR 可以克服这个问题。后两个实验表明，吖啶橙处理与两步嵌套 PCR 相结合的方法对于微生物生态学中活细胞的检测是有用的。
