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应用PCR方法检定单核细胞增多性李斯特菌 |
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姜永强1 雷祚荣1 李瑾2 马颖2
【摘要】 目的 建立单核细胞增多性李斯特菌(单增李斯特菌)快速、敏感、
特异的PCR诊断方法。方法 选取hlyA基因作为靶序列设计一对引物，用该引物
对54株标准李斯特菌和21株无关菌进行PCR扩增，得到进一步验证。采用PCR和
常规鉴定方法同时对从国内食品分离的33株李斯特菌进行鉴定。结果 可扩增含
有保守Hind Ⅲ切点的743 bp片段。表现出极好的单增李斯特菌种特异性。纯培养
的检测极限为55个菌。发现其中18株用两种方法都鉴定为单增李斯特菌，两种方
法的结果完全符合。当用于食物标本检测时，PCR反应遭强烈抑制。经过25小时
的增菌培养，对培养物进行化学抽提、纯化的菌体经加热裂解后直接用作PCR反
应模板，可使抑制作用大大降低。每毫升牛奶人工接种4个菌可用该法特异检出。
结论 建立了PCR方法，可初步用于单增李斯特菌的快速、特异、敏感诊断。
【关键词】 单核细胞增多性李斯特斯特菌 聚合酶链反应
Identification of Listeria monocytogenes by PCR Method Jiang Yongqiang*, Lei Zuorong,
Li Jin, et al. *Institute of Microbiology and Epidemiology, Academy of Military Medical
Sciences, Beijing 100071
【Abstract】 Objective To establish a rapid, sensitive and specific polymerase chain
reaction (PCR) method for detection of Listeria monocytogenes (LM). Methods A pair of
oligonucleotide primers were designed with hylA gene as target sequence. Fiftyfour strain of
standard LM and 21 strains of irrelevant bacteria were amplified and confirmed by PCR
technique. Thirty-three strains of listeria isolated from food at home were identified by PCR
and routine methods. Results A 743-bp DNA fragment with a conservative HindⅢ site
could be amplified by PCR, showing excellent features of LM. Limit to detection for pure
culture was 55 colony forming units (CFU). Eighteen of 54 strains were identified as LM by
both methods, with a completely coincident result. PCR was strongly inhibited as used in
detection for food. If food was cultured for 25 hours for proliferation in listeria enrichment
broth, then the culture was chemically extracted, the purified bacteria after heat lysis could
react as PCR templates and the inhibitory effects could be greatly reduced. Milk inoculated
artificially with four colony forming units of LM could be specifically detected by PCR.
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