单核细胞增生性李斯特菌PCR快速检测方法建立及应用  

Rapid detection of Listeria monocytogenes by PCR

作　　者：巢国祥 徐勤 周晓辉[2] 唐丽华[2] 焦新安[2] 

机构地区：[1]扬州市疾病预防控制中心,扬州225002 [2]扬州大学生物科学与技术学院

出　　处：《中国人兽共患病杂志》2004年第9期797-800,共4页Chinese Journal of Zoonoses

基　　金：教育部"高校青年教师奖"(175 );"江苏省六大人才高峰"资助项目(G -2 0 0 2 -0 2 6);江苏省"十五"科技攻关项目(招标项目 16)

摘　　要：目的　通过扩增hly基因PCR法建立快速检测单核细胞增生性李斯特氏菌 (Lm )方法。 方法 :用PCR法扩增hly基因来检测标准菌株及临床分离的Lm的纯培养物和模拟污染的生猪肉、水和牛奶 ,并应用于实际食品检验工作中进行验证。结果　 34株Lm的PCR结果呈阳性 ,而其它李斯特菌 ,包括英诺克李斯特菌、绵羊李斯特菌、西尔李斯特菌、威儿李斯特菌和格氏李斯特菌以及非李斯特菌均未出现特异性的片段。表明所扩增的hly基因 3’末段 82 7bp的DNA片段对Lm具有较高的特异性。PCR法对Lm纯培养物的检测限达 10 5CFU/ml,对模拟污染生猪肉、水和牛奶的 30℃ 16h改良LEB增菌培养液用PCR法检测 ,结果检测限达 10CFU/ 2 5 g(ml)。进一步研究表明该PCR扩增体系能检测出 6 .5pg的LmDNA ,整个检测过程只需 2 4h。采用该法对扬州市区 16 9份食品样品检测 ,8份样品Lm呈阳性且结果与传统的生化鉴定方法完全一致。结论　扩增hly基因的PCR法对Lm的鉴定具有特异性强、灵敏度高、速度快和易操作等优点 。To establish a rapid method by amplification of the hly gene of this organism with PCR for the detection of Listeria monocytogenes in pure culture, and mimic contaminated water; milk and raw pork and for the application of this method in the routine food examination,it was found that the PCR products could be detected in 34 strains of L.monocytogenes, but not found in other Listeria spp, including L.innocua, L.ivanovii, L.seelgeri, L.welshimeri, L.grayi, and also not found in other non Listeria species.These results indicate the primer set (827 bp fragment at hly 3' end) was highly specific for L.monocytogenes. The detection limit of this PCR assay was 10 5 CFU/ml of pure cell culture. However, this method of assay could detect as few as 10 CFU of L.monocytogenes in the mimic contaminated pork (25 g) or contaminated water and milk(25ml), at 30℃ for 16 hours,or in LEB medium at 30℃. Further study showed this PCR assay could detect as few as 6.5 pg DNA of L.monocytogenes and the total assay time was approximately 24 hours.From 169 food samples in Yangzhou area, 8 samples were proved to be positive for L.monocytogenes by means of this method of assay, and the results were quite consistent with those detected by conventional biochemical testings. It is concluded this method of PCR assay is highly sensitive, specific and easy to handle, and can be used as a rapid detection method to find out L.monocytogenes in water, milk, pork and other food products.

关 键 词：单核细胞增生性李斯特菌(Lm) 快速检测 PCR 

分 类 号：R378.99[医药卫生—病原生物学]