摘要
【目的】通过对检测食源性致病菌单核细胞增生性李斯特氏菌(LM)的多重PCR体系的优化,建立一种能够在食品样品中应用的四重PCR。【方法】采用热裂解提取菌液和菌落DNA,以16SrRNA、inlAB、iap、hly基因为靶基因设计引物,从影响多重PCR扩增的引物浓度、退火温度进行优化。【结果】通过其它5种同属异种菌即英诺克李斯特氏菌、西尔李斯特氏菌、威尔西李斯特氏菌、绵羊李斯特氏菌、格氏李斯特氏菌及副溶血性弧菌均未扩增出特异性的片段。纯培养物的最低检测限为102CFU/mL,模拟污染的生猪肉检测限不大于0.4CFU/g。【结论】通过菌落获得DNA的多重PCR方法更好,该方法具有敏感、特异、快速及准确的优点,可用于食品中LM的快速检测。
【Objective】 A multiple polymerase chain reaction（PCR） system was developed to detect Listeria monocytogenes（LM）,the more important food born pathogens,and applied to detect LM from food samples.【Method】 Template DNA was obtained by thermolysis broth cultured bacteria and thermolysis colony.Primers were designed according to the 16S rRNA,inlAB,iap,hly genes.The concentration of primers and annealing temperature were examined and optimized.【Result】 The PCR products were not detected in other Listeria spp,including L.innocua,L.seeligeri,L.welshimeri,L.ivanovii,L.grayi and in Vibrio parahemolyticus,indicating that this method was highly specific for L.monocytogenes.The detection limit of the PCR assay was 102 CFU/mL of pure cell culture.The PCR assay could detect no more than 0.4 CFU/g of L.monocytogenes in the contaminated pork.【Conclusion】 The results show that the colony PCR method is better,the method assay is highly sensitive,specific,rapid and accurate and can be used for rapid detection of L.monocytogenes in food.
出处
《中国农业科学》
CAS
CSCD
北大核心
2010年第23期4893-4900,共8页
Scientia Agricultura Sinica
基金
上海市科技兴农重点攻关项目(沪农科攻字2005第4-2号
2009第6-1号)
上海市教育委员会重点学科建设项目(J50704)
上海海洋大学骆肇荛大学生科技创新基金项目(G-7101-00-000712)
作者简介
刘海泉，博士研究生。Tel：15121039982：E-mail：haiquanl917@gmail．com。
通信作者赵勇，副教授。Tel：021-61900379：Fax：021-61900379；E-mail：yzhao@shou．edu．cn
