实时荧光定量PCR方法应用于药品无菌快速检测的研究

Real-time Fluorescence PCR Assay for Fast Detection of Drug Sterility Test

关键词:  荧光定量PCR  叠氮溴化丙锭  细菌污染  快速检测

Key words:  real-time fluorescence quantitative PCR  propidium monoazide (PMA)  bacterial contamination  fast detection

摘要:

目的 建立应用实时荧光定量PCR进行无菌快速检测的方法。方法 选取金黄色葡萄球菌及大肠埃希菌，用裂解试剂盒抽提细菌基因组DNA，进行实时荧光定量PCR检测，并应用叠氮溴化丙锭(PMA)抑制样品中死菌基因组DNA的PCR扩增。结果 PMA能有效去除样品中死菌干扰，针对16S rRNA基因保守序列进行扩增的荧光定量PCR方法具有较高的灵敏度。在金黄色葡萄球菌和大肠埃希氏菌检测中，最低含菌量组与阴性对照组Ct值有明显差异(P<0.05)，其最低检出限为2 CFU/PCR。在对人工污染药品的无菌检测中，该方法与药典检测方法结果一致。结论 进行无菌检查时，采用PMA去除样品中死菌基因组DNA干扰，以裂解试剂盒抽提细菌基因组后用荧光定量PCR分析样品中细菌污染，可将检测时间缩短到4 h左右，操作简单，灵敏性高，可应用于药品无菌检查的快速筛查。

Abstract:

OBJECTIVE To establish a real-time PCR assay to fast detect bacterial contamination in drugs. METHODS Staphylococcus aureaus and Escherichia coli were used as representative, and the bacterial genomic DNA was isolated by lysis buffer kit and then quantitatively detected by Taqman real-time PCR. Propidium monoazide (PMA) was used as a pretreatment for the DNA molecules in dead cells and inhibited the PCR amplification of the DNA molecules. RESULTS PMA treatment was effective and the real-time PCR targeting conservative 16S rRNA gene showed high sensitivity. The Ct value of the lowest bacterial dose groups in both S. aureaus and E. coli were statistically different from the negative control with a minimal detection quality of 2 CFU/PCR. The result of real-time PCR was accordant to the pharmacopoeia test in the sterile test of contaminated drugs. CONCLUSION The lysis buffer kit used to extract bacterial genomic DNA with subsequent real-time PCR is convenient and sensitive, and may be applied to fast detection of drug sterility test.