鼠伤寒沙门氏菌PCR快速检测试剂盒的研制  

Development a novel PCR kit for the rapid detection of Salmonella typhimurium

作　　者：侯宛宛 李可 张艺沛 方莹 陈晴 柳雅馨 刘斌[1,2] 岳田利[1] HOU Wanwan;LI Ke;ZHANG Yipei;FANG Ying;CHEN Qing;LIU Yaxin;LIU Bin;YUE Tianli(College of Food Science and Engineering,Northwest A & F University,Yangling 712100;Fuping Modern Agriculture Comprehensive Demonstration station,Northwest A & F University,Fuping 711799;Zhejiang Academy of Science & Technology for Inspection & Quarantine,Hangzhou 310016)

机构地区：[1]西北农林科技大学食品科学与工程学院,杨凌712100 [2]西北农林科技大学,富平现代农业综合试验示范站,富平711799 [3]浙江省检验检疫科学技术研究院,杭州310016

出　　处：《食品科技》2019年第7期359-366,共8页Food Science and Technology

基　　金：国家重点研发计划项目(2018YFC1602201);国家自然基金项目(31471638);浙江省公益性技术应用研究(分析测试)项目(2018C37015);质检总局科技计划项目(2017IK206);西北农林科技大学试验示范站(基地)科技成果推广项目(TGZX2017-27)

摘　　要：为了能快速、准确、灵敏地检测鼠伤寒沙门氏菌,而研制一种基于PCR技术的检测试剂盒。该试剂盒采用了一对特异引物,该引物是以鼠伤寒沙门氏菌的基因STM4495中一段特异序列为检测靶点设计的,可使试剂盒具有良好的特异性。特异性评价试验结果显示,可从22株沙门氏菌(12种血清型)和20株非沙门氏菌(12个常见属)中,准确地检测出鼠伤寒沙门氏菌。此外,从5种PCR增强剂中筛选到了3种,并添加到试剂盒中,以避免PCR反应受到引物二聚体和食品中PCR抑制剂的干扰,使试剂盒具备良好的灵敏性。经评价试验表明,试剂盒检测鼠伤寒沙门氏菌的检测限可以达到37.5fg/PCR,即使贮存于—20℃放置30d,仍然能够达到原有检测限;而将该试剂盒反复冻融60次后,也可达375fg/PCR。对食品样品进行检测,经过增菌24h后,可以从0~1CFU/10g乳粉中检测出鼠伤寒沙门氏菌。从以上结果可知,研制的PCR试剂盒能够准确地检测出鼠伤寒沙门氏菌,具有较高的灵敏性和稳定性,可以用于食品中鼠伤寒沙门氏菌的快速筛检。To detect Salmonella typhimurium rapidly,accurately and sensitively,a novel PCR kit had been developed in this study.For improving accuracy of PCR kit,a primer set was designed based on a specific fragment from STM4495 gene of Salmonella typhimurium.The result of specificity test showed that the PCR kit could detect out Salmonella typhimurium from 22 Salmonella strains,including 12 serotypes,and 20 non-Salmonella strains belonged to 12 genus.Moreover,3 additives were selected from 5 enhancers,added into the PCR kit to avoid interference of primer dimers and PCR inhibitor,which could help to improve sensitivity of the PCR kit.According to the result of sensitivity test,the detection limit of the PCR kit was 37.5 fg/PCR for purified genomic DNA of Salmonella typhimurium ATCC14028.The detection limit didn’t change when stored at-20℃for 30 days,or only rose high to 375 fg/PCR after frozen-thawed for 60 times.For detection of artificial contamination milk powder,the samples could be detected by the PCR kit after 24 h enrichment when 0~1 CFU of Salmonella typhimurium ATCC14028 strain was inoculated in 10 g milk powder.These results indicated that the PCR kit was sensitivity,stability and accuracy,which could be applied into food detection of Salmonella typhimurium.

关 键 词：鼠伤寒沙门氏菌 聚合酶链式反应(PCR) 检测试剂盒 

分 类 号：TS207.4[轻工技术与工程—食品科学]