应用RCP方法检定单核细胞增多性李斯特菌

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７４３ｂｐ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

Identification of Listeria monocytogenes by PCR method]

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 irelevant 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 Hind II 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. CONCLUSION: PCR can be used in rapid, sensitive and specific identification of LM.