基于ttrA基因优化环介导等温扩增技术检测食品中沙门氏菌  

Detection of Salmonella in Food Using ttrA Gene Optimized Loop-Mediated Isothermal Amplification Technology

作　　者：任翔 邹颜秋硕 汤晓召 杨菁 杨祖顺[1] REN Xiang;ZOU Yanqiushuo;TANG Xiaozhao;YANG Jing;YANG Zushun(Yunnan Provincial Center for Disease Control and Prevention,Kunming 650022,China)

机构地区：[1]云南省疾病预防控制中心,云南昆明650022

出　　处：《食品科技》2024年第4期353-360,共8页Food Science and Technology

基　　金：云南省重大科技专项(202002AE32005)。

摘　　要：目的:优化食品沙门氏菌环介导等温扩增检测技术(Loop-mediated isothermal amplification,LAMP),更快更准确地检测食品中沙门氏菌,为食品中沙门氏菌的防控提供技术支撑。方法:根据沙门氏菌ttrRSBCA转座子中ttrA四亚硫酸还原酶亚基A基因,重新设计新的环介导等温扩增检测引物,通过与发表的invA1引物、invA2引物、invA3引物、bcfD引物及市售两款D牌及S牌检测试剂盒进行范围性实验、特异性实验、灵敏度实验,对该引物的LAMP检测方法进行验证。结果:重新设计的ttrA、ttrB-2、ttrA120-1引物在检测沙门氏菌方面具有较强的稳定性。在对14种非沙门氏菌检测中,引物ttrA120-1、引物bcfD和市售S牌检测试剂盒排他性达到100%。引物ttrA及市售D牌检测试剂盒排他性分别为64.3%和85.7%。invA1、invA3引物排他性为0%,特异性极差。在对26种不同血清型沙门氏菌检测时,引物ttrA120-1能有效检测23种沙门氏菌,检测包容性为88.5%。引物ttrA能有效检测22种沙门氏菌,包容性为84.6%。引物bcfD能够检测出19种沙门氏菌,包容性为73.1%。市售的D牌及S牌检测试剂盒分别能检测19种及20种沙门氏菌,包容性分别为73.1%和76.9%。灵敏度实验中,DNA浓度在3.2×10^(–5) ng/μL时,使用ttrA120-1引物可以正常检测。结论:引物ttrA120-1在灵敏性、特异性、检测能力上都优于文章所对比的市售试剂盒及所发表的引物序列,能够更准确、更高效地在食品沙门氏菌快速检测中进行运用。Objective:The objective of this research is to optimize the loop-mediated isothermal amplification(LAMP)detection technology for faster and more accurate detection of Salmonella which could provide the technical support for the prevention and control in food.Methods:A redesigned LAMP detection primer was developed based on the ttrA gene found in the ttrRSBCA transposon of Salmonella,the primer's were validated through experiments testing its range,specificity,and sensitivity,and it was compared to invA1,invA2,invA3,and bcfD primers,as well as two commercially available D and S brand detection kits.Results:The redesigned primers ttrA,ttrB-2,and ttrA120-1 demonstrated strong stability in detecting Salmonella.Additionally,primers ttrA120-1,bcfD,and a commercially available S brand detection kit exhibited 100%exclusivity for 14 non-Salmonella species in this detection.And the exdusivity of primer ttrA and commercially available D test kits was 64.7%and 85.7%.The primers of invA1,invA3 exhibited 0%exclusivity and poor specificity.The primer ttrA120-1 could effectively detect 23 Salmonella species across 26 different serotypes with an inclusiveness rate of 88.5%.Similarly,the primer ttrA can detect 22 species with an inclusiveness rate of 84.6%.The primer bcfD had a detection inclusiveness rate of 73.1%for 19 species.Commercially available D and S test kits could detect 19 and 20 Salmonella species with inclusiveness rates of 73.1%and 76.9%.The ttrA120-1 primer was able to detect DNA concentrations as low as 3.2×10^(–5)ng/μL in sensitivity experiments.Conclusion:The primer ttrA120-1 has higher sensitivity,specificity,and detection ability compared to commercially available kits and published primer sequences,making it a more accurate and efficient tool for the rapid detection of Salmonella in food.

关 键 词：食源性疾病 沙门氏菌 ttrA基因 环介导等温扩增 引物 

分 类 号：TS207.4[轻工技术与工程—食品科学]