应用环介导等温扩增技术快速检测单核细胞增生李斯特菌的研究

目的建立环介导等温扩增技术快速检测单核细胞增生李斯特菌。方法根据单核细胞增生李斯特菌(LM)hlyA基因序列中的保守区域,采用在线引物设计软件Primer Explorer4.0进行设计,获得一套特异性的环介导等温扩增(LAMP)引物,对单核细胞增生李斯特菌hlyA基因进行LAMP扩增,并与常规PCR方法进行比较。结果建立的LAMP方法能成功扩增出梯形条带,LAMP检测单核细胞增生李斯特菌纯培养物和人工染菌的灵敏度为5.44×102cfu/mL,而对照PCR检测的灵敏度为5.44×104cfu/mL。对10株细菌进行LAMP扩增,仅单核细胞增生李斯特菌得到阳性结果。从DNA提取到报告结果,耗时仅1h。结论 LAMP检测单核细胞增生李斯特菌灵敏度高,特异性强,耗时短,方法简便,有望发展成为快速检测食品中单核细胞增生李斯特菌的有效手段。

Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Listeria Monocytogenes

According to GenBank-published Listeria monocytogenes hlyA gene sequence,three pairs of specific loop-mediated isothermal amplification primers were designed and a novel and highly specific loop-mediated isothermal amplification assay was developed for sensitive and rapid detection of Listeria monocytogenes. The assay correctly identified Listeria monocytogenes,but did not detect 9 non-Listeria monocytogenes strains.Sensitivity of the LAMP asssay for direct detection of Listeria monocytogenes in pure cultures and in chicken meat samples was 2.0 CFU per reaction. The sensitivity of the LAMP assay was 100-fold more than that of the conventional PCR assay. The LAMP assay for detecion of Listeria monocytogenes required 60 min in chicken meat samples from the beginning of DNA extraction to final determination. The LAMP assay is a sensitive,rapid and simple tool for the detection of Listeria monocytogenes and will facilitate the surveillance and control of Listeria monocytogenes contamination in food.