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ＨｉｎｄⅢ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。
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. Conclusion PCR can be used in rapid, sensitive and specific identification of LM .
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
1998年第1期19-21,共3页
Chinese Journal of Preventive Medicine
