摘要
为实现多种致病性大肠埃希氏菌和沙门氏菌及其耐药基因的快速、准确检测,建立一种微流体芯片检测方法。通过筛选肠致病性大肠埃希氏菌、产肠毒素大肠埃希氏菌、肠出血性大肠埃希氏菌、肠炎沙门氏菌、鼠伤寒沙门氏菌、鸡白痢沙门氏菌和鸡伤寒沙门氏菌的特异性基因作为病原鉴定基因,以喹诺酮类、氨基糖苷类、四环素类和氯霉素类4类抗菌药物的13个耐药基因作为耐药检测靶基因,针对12个病原鉴定基因和13个耐药基因,设计25对特异性引物,并在每个正向引物5′端添加通用标签序列;通过引物筛选验证、扩增程序优化、特异性试验、灵敏度试验、重复性试验和临床样品检测,建立了可同时鉴别检测多种致病性大肠埃希氏菌和沙门氏菌及其耐药基因的微流体芯片检测方法。结果显示,25个目标基因与其他病原或基因之间均无交叉反应,特异性好;25个基因的检测灵敏度在0.004~40 pg/μL之间,灵敏度高;重复检测稳定性好。建立的微流体芯片检测方法可用于同时进行多种致病性大肠埃希氏菌和沙门氏菌及其耐药基因的鉴别检测,具有快速、特异、敏感、稳定等优点,为动物疾病诊断和流行病学调查提供了有效途径。
To achieve rapid and accurate detection of various pathogenic Escherichia coli,Salmonella and antimicrobial resistance genes,a microfluidic chip detection method was established.The specific genes of enteropathogenic Escherichia coli,enterotoxigenic Escherichia coli,enterohemorrhagic Escherichia coli,Salmonella enteritidis,Salmonella typhimurium,Salmonella pullorum and Salmonella gallinarum were screened as pathogen identification genes,and 13 resistance genes from four classes of antimicrobials including quinolones,aminoglycosides,tetracyclines and chloramphenicols were selected as resistance detection target genes,25 pairs of specific primers were designed for 12 pathogen identification genes and 13 antimicrobial resistance genes,and a universal tag sequence was added to the 5′end of each forward primer.Through primers screening and verification,the amplification procedure optimization,specificity testing,sensitivity testing,repeatability testing and clinical sample detection,a microfluidic chip detection method was established to simultaneously identify and detect various pathogenic Escherichia coli,Salmonella and antimicrobial resistance genes.The results showed that the 25 target genes had no cross-reactivity with other pathogens or genes,demonstrating excellent specificity.The detection sensitivities of the 25 genes ranged from 0.004 to 40 pg/μL,indicating high sensitivity,and the repeated tests exhibited good stability.The microfluidic chip detection method developed in this study could be used for simultaneous identification and detection of various pathogenic Escherichia coli,Salmonella and antimicrobial resistance genes.It had the advantages of rapidity,specificity,sensitivity and stability,providing an effective approach for animal disease diagnosis and epidemiological investigation.
作者
傅彩霞
刘海莹
栗云鹏
张玮
周德刚
张启龙
FU Cai-xia;LIU Hai-ying;LI Yun-peng;ZHANG Wei;ZHOU De-gang;ZHANG Qi-long(Beijing Center for Animal Disease Prevention and Control,Beijing 102629,China)
出处
《动物医学进展》
北大核心
2025年第10期76-84,共9页
Progress In Veterinary Medicine
基金
北京市农业科技项目(20180123)。
关键词
致病性大肠埃希氏菌
沙门氏菌
耐药基因
微流体芯片
Pathogenic Escherichia coli
Salmonella
antimicrobial resistance gene
microfluidic chip
作者简介
傅彩霞(1971-),女,新疆博乐人,硕士,正高级兽医师,主要从事动物疫病检测及防治技术研究推广;通讯作者:周德刚;通讯作者:张启龙。
