摘要
建立有效区分死菌、活菌PCR检测方法,用于药品中细菌污染检测。选取金黄色葡萄球菌及大肠埃希菌,用叠氮溴化丙锭(PMA)前处理,使PMA与死菌DNA分子共价交联,抑制该DNA分子PCR扩增。当PMA浓度大于5μg·mL-1、曝光时间大于5 min时,PMA可抑制细胞膜破裂革兰氏阴性、阳性菌死菌DNAPCR扩增;PMA浓度大于20μg·mL-1时对活菌DNAPCR扩增产生一定影响。经过PMA前处理,能有效抑制死菌DNA扩增,在药品细菌污染PCR检测中有很好应用前景。
To establish a available PCR assay of differentiate dead cells from viable ones for detecting bacterial contamination in drugs. Staphylococcus aureaus and Escherichia coil were used as representative, propidium monoazide （PMA） was used as a pretreatment for the genome extraction, in which PMA covalently crosslink with DNA molecules in dead cells and inhibit the PCR amplification of DNA molecules. The results indicate that the PCR amplification of DNA from dead gram-negative bacterium and gram-positive bacterium cell can be inhibited by PMA with a mass concentration of 5 μg .mL-1 after an exposure for more than 5 rain, while the PCR amplification of DNA from viable cells is inhibited by PMA with concentration of more than 20μg. mL-1. Propidium monoazide （PMA） pretreatment can effectively inhibit the PCR amplification of DNA molecules from dead cell.
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2013年第9期91-95,共5页
Journal of Northeast Agricultural University
基金
广东省科技计划项目(2011B031500029)
