饲料中肠炎沙门氏菌CPA-LFD检测方法的建立  

Establishment of CPA-LFD detection method for Salmonella enteritidis in feed

作　　者：潘展锋 车立忱[2] 齐景文[2] 杜润东 袁帅 周铁忠 唐峰 PAN Zhan-feng;CHE Li-chen;QI Jing-wen;DU Run-dong;YUAN Shuai;ZHOU Tie-zhong;TANG Feng

机构地区：[1]锦州医科大学畜牧兽医学院,辽宁锦州121000 [2]沈阳市动物疫病预防控制中心,辽宁沈阳110000 [3]康平县现代农业服务中心,辽宁沈阳110500 [4]锦州市现代服务学校,辽宁锦州121011

出　　处：《饲料研究》2022年第7期109-112,共4页Feed Research

基　　金：辽宁省科技厅自然科学基金指导计划项目(项目编号:20180551177);辽宁省科技厅项目(项目编号:2020JH1/10200003);辽宁省教育厅项目(项目编号:202028-59)。

摘　　要：为建立饲料中肠炎沙门氏菌(Salmonella enteritidis,SE)的快速检测方法,针对sdfⅠ基因设计交叉引物,优化反应条件,建立饲料中肠炎沙门氏菌的交叉引物等温扩增联合横向流动试纸条可视化检测法(CPALFD)。检测条件:反应时间60 min、反应温度63℃、甜菜碱浓度1.2 mmol/L、Mg^(2+)浓度5.0 mmol/L、BST DNA聚合酶8 U、dNTPs浓度0.3 mmol/L。结果显示:检测限为1.0×10^(1)ng/L,与鼠伤寒沙门氏菌、鸡白痢沙门氏菌、猪霍乱沙门氏菌、德尔卑沙门氏菌、单增李斯特菌、志贺氏菌等常见菌无交叉反应;对56份疑似污染样品检测结果显示,CPA-LFD法与国标法和PCR法检测结果一致。研究表明,初步建立饲料中肠炎沙门氏菌的CPA-LFD检测方法。To establish a rapid detection method for Salmonella enteritidis(SE) in feed, cross priming amplification(CPA) primers were designed for sdf Ⅰ gene, and reaction conditions were optimized. A novel and rapid cross priming amplification(CPA) method combined with a lateral flow dipstick(LFD) were developed. The optimal detection conditions were as follows: reaction time 60 min, reaction temperature 63 ℃, betaine concentration 1.2 mmol/L, Mg^(2+) concentration 5.0 mmol/L, BST DNA polymerase 8 U, dNTPs concentration 0.3 mmol/L. The results showed that the detection limit was 1.0×10^(1) ng/L, which had no cross-reaction with common bacteria such as Salmonella typhimurium,Salmonella pullorum, Salmonella choleraesuis, Salmonella delpy, Listeria monocytogenes and Shigella. The test results of56 suspected contaminated samples showed that the CPA-LFD method was consistent with the national standard method and PCR method. The experiment indicates that a CPA-LFD detection method for Salmonella enteritidis in feed is initially established.

关 键 词：肠炎沙门氏菌 饲料 交叉引物扩增 可视化核酸检测 

分 类 号：S816.17[农业科学—饲料科学]