小鼠冷冻胚胎、精子常见病原菌快速检测方法的建立

摘要

Objective To establish a rapid monitoring method of the three common bacteria in mice frozen resources, such as embryo, sperm, etc. Methods To extract DNA of the three positive bacteria( Staphylococcus auerus, Klebsiella⁃pneumoniae and β⁃hemolyticstreptococcus) , and establish PCR monitoring method of the three positive strains through designing primer and refining PCR condition. Then extract total DNA of the frozen resources, detect the DNA according to the PCR condition of the three positive bacteria, some samples were detect by fluorescence quantitative PCR at the same time. Results ①successfully establish a PCR detection method of the three positive bacteria, the minimum detectable concentration of Staphylococcus auerus, Klebsiella pneumoniae and β⁃hemolytic streptococcus is 4�19 × 10 -5 ng/μL, 1�98 × 10 -5 ng/μL and 1�07 × 10 -3 ng/μL. ②Proved that the three bacteria doesn ’ t exist in the sample by normal PCR and fluorescence quantitative PCR methods. Conclusions Establising a rapid monitoring method of common pathogens of frozen embryo and sperm in mice.%目的：建立实验动物冷冻资源（胚胎、精子等）常见病原菌的微量、快速监测方法。方法以实验动物三个常见病原菌（金黄色葡萄球菌、肺炎克雷伯氏菌和乙型溶血性链球菌）为研究对象，分别提取三个阳性菌株的DNA，通过引物设计和相应PCR条件的优化，建立三种阳性菌株的PCR检测方法。然后提取冷冻资源（胚胎、精子等）中的DNA，按照三个阳性菌的PCR条件进行检测，并将一部分样品DNA用荧光定量PCR方法进行检测。结果①成功建立三个阳性菌株的PCR检测方法。三种病原菌的最低检测浓度，金黄色葡萄球菌为4�19×10－5 ng／μL，肺炎克雷伯氏菌为1�98×10－5 ng／μL，乙型溶血性链球菌为1�07×10－3 ng／μL．②通过普通PCR和荧光定量PCR方法均未检测出样品中三种阳性菌的存在。结论建立了小鼠冷冻胚胎、精子常见病原菌微量、快速检测方法。