Dole qPCR检测嗜肺巴斯德杆菌的可靠性研究及在啮齿类实验动物质量监测中的应用

目的 研究适用于啮齿类实验动物质量监测中嗜肺巴斯德杆菌快速可靠的检测方法。方法 对17株标准菌株及76株嗜肺巴斯德杆菌疑似分离菌株进行检测,分析比较文献中4种PCR检测方法的敏感度和特异度,选择最优PCR方法,并将其应用于22个厂家的312只实验动物(包括191只小鼠,57只大鼠,64只沙鼠)气管和肠内容物样本的检测,同时与国标中的细菌分离培养加生化鉴定方法检出率进行比对。结果 Dole qPCR和Benga mPCR能特异检出疑似嗜肺巴斯德杆菌的分离株中所有的Heyl型和Jawetz型; Benga mPCR和巴斯德菌科引物的Bootz PCR虽能检出疑似分离菌株中所有嗜肺巴斯德杆菌Heyl型和Jawetz型,却不能与巴斯德菌科其它细菌相区分; 高正琴等^[8]报道的qPCR方法,虽然敏感度高,但特异度较差。因此选择了敏感度高、特异度强的Dole qPCR方法,应用于啮齿类实验动物呼吸道与肠内容物样本嗜肺巴斯德杆菌的筛查。对191只小鼠,57只大鼠以及64只沙鼠调查显示,小鼠气管和肠内容物中阳性率分别为25.7%和27.2%,大鼠分别为28.1%和29.8%,沙鼠分别为26.6%和21.9%; 小鼠、大鼠和沙鼠的PCR总阳性率(气管和/或肠内容物阳性)分别为39.3%,35.1%和34.4%,而传统分离培养方法总检出率仅为4.7%,15.8%和23.4%。结论 嗜肺巴斯德杆菌DoleqPCR方法的阳性检出率远高于传统培养方法,更适合于实验动物的快速质量监测。

Reliability Research of Dole qPCR Method on Pasteurella Pneumotropicaand Application in Health Screening of Laboratory Rodents

Objective To study the rapid and reliable detection method for Pasteurella pneumotropica(PP)and apply it to the quality monitoring of laboratory rodents.Methods To compare the sensitivity and specificity of 4 different polymerase chain reaction(PCR)methodson 17 standard strains and 76 suspected isolates of PP and select the best PCRmethod,tacheas and intestinal contents from 312 rodent experimental animal(191mice,57 rats,64 gerbils)were chosen to compare the detection rates of PCR and national standard bacterial isolation culture plus biochemical identificationmethods.Results Dole qPCR and Benga Multiplex PCR(mPCR)could detect Pasteurella pneumotropica specifically,but the former was much more sensitive than the other.Both Benga mPCR and Bootz PCR could detect allHeyl and Jawetz types in suspected isolates,but not distinguish them from otherbacteria in Pasteuraceae.Gao's qPCR had a high sensitivity but lack of specificity.Therefore,Dole qPCR was selected to test PP health screening samples,and the positive rate in tracheas and intestinal contents of mice were 25.7% and27.2%,28.1% and 29.8% in rats,26.6% and 21.9% in gerbils respectively.The total PCR positive rates(tracheas and/or intestinal contents)in mice,rats and gerbils were 39.3%,35.1% and 34.4% respectively.While the positive ratesof traditional cultivation method were only 4.7%,15.8% and 23.4% respectively.Conclusion With far higher sensitivity and specificity than other reported PCR methods and biochemical identification method,the Dole's qPCR is more suitable for the health screening of PP in laboratory rodents.