Development of triplex TaqMan MGB-probe real-time PCR assay for detection of Listeria monocytogenes and the distribution of virulence gene in isolates
LIU Er-long1, YUAN Mu-yun2, LU Ying-zi1, CHEN Ying3, WANG Ping3, XU Long-Yan2, LI Jia-qi1, ZHENG Gao-bing1, DU Ya-ping1, LIN Xue-qin1
1.Huangpu Entry-Exit Inspection and Quarantine Bureau, Guangzhou 510730, China; 2. Guangdong Entry-Exit Inspection and Quarantine Bureau, Guangzhou 510730, China; 3.Chinese Academy of Inspection and Quarantine, Beijing 100025, China
Abstract：To develop a sensitive and specific triplex TaqMan-MGB real-time PCR assay for the detection of Listeria monocytogenes isolated from animal derived food,specific primers and MGB probes targeted toward the hylA,inlA and plcB gene were designed based on the conserved sequences.Then,the virulence genes distribution among groups(groupI,groupII,groupIII) which aligned by Multilocus sequence typing (MLST)was analyzed. The results showed that the assay developed in this study has good sensitivity,specificity and repeatability for the detection of Listeria monocytogenes.The detection limit was 100 CFU/mL in pure culture.and the amplifying efficiencies and the correlation coefficient of standard curves were all in the acceptable ange.The positive rates of hylA,inlA and plcB were 98% (99/101),75% (76/101) and 98% (99/101) respectively in 101 Listeria monocytogenes strains. Among 89 of 101 strains classed into 3 clusters(groupI,groupII and groupIII) by MLST,20 of 30 isolates belonged to groupI were all negative in inlA gene;56 of 57 isolates belonged to groupII were positive in all 3 virulence gene;2 isolates grouped into groupIII which hlyA,inlA and plcB were all negative.The detection result showed that the distrubution of 3 virulence gene consistented with the type groups clustered by MLST and provided remarkable references value for evaluating the virulence of Listeria monocytogenes.
刘二龙, 袁慕云, 吕英姿, 陈颖, 王娉, 许龙岩, 李嘉琪, 郑高彬, 杜雅萍, 林学勤. 单增李斯特菌三重实时荧光PCR检测的建立及其毒力基因在分离菌株中分布[J]. 中国人兽共患病学报, 2016, 32(5): 451-456.
LIU Er-long, YUAN Mu-yun, LU Ying-zi, CHEN Ying, WANG Ping, XU Long-Yan, LI Jia-qi, ZHENG Gao-bing, DU Ya-ping, LIN Xue-qin. Development of triplex TaqMan MGB-probe real-time PCR assay for detection of Listeria monocytogenes and the distribution of virulence gene in isolates. Chinese Journal of Zoonoses, 2016, 32(5): 451-456.
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2016, Vol. 32 
