沙门菌tHDA等温扩增检测方法的建立

Establishment of tHDA assay for the detection of salmonella

目的建立基于依赖耐热解旋酶核酸等温扩增(t HDA)的沙门菌快速检测方法。方法从Gen Bank数据库下载沙门菌侵袭蛋白A(inv A)基因,比对确定保守区并设计t HDA引物,并对引物浓度、硫酸镁以及氯化钠浓度等反应条件进行优化。同时评价方法的特异性和灵敏度,并检测模拟样本和真实样本以验证方法的可靠性。结果建立的沙门菌t HDA检测体系经过优化,结果显示仅沙门菌菌株呈现特异性电泳条带,而志贺菌、副溶血弧菌、金黄色葡萄球菌、大肠杆菌O157∶H7、霍乱弧菌等其他5种肠道细菌均为阴性,特异性好;t HDA法检测沙门菌纯培养物的灵敏度为19cfu/μl,检测模拟样本的灵敏度为260 cfu/μl。采用t HDA方法检测18份真实样本,共检出9份沙门菌阳性,与国标法检测结果一致。结论沙门菌t HDA检测方法快速、灵敏、特异,在食源性疾病检测及监测中具有重要的技术优势。

Objective To establish thermophilic helicase-dependent amplification（t HDA） assay for Salmonella detection.Methods With the invasive protein A（inv A） gene of Salmonella strains download from Gene Bank,the conserved region was identified to design t HDA primers.The primer and Mg SO4,Na Cl concentration were optimized.The specificity and sensitivity of the assay were evaluated.The simulated and real samples were used to verify the reliability of the assay.Results By optimization,it was showed that only Salmonella strain showed specific electrophoreisis band,while other 5 kinds of enteric pathogen strain,Shigella,Vibrio parahaemolyticus,Staphylococcus aureus,Escherichia coli O157：H7,Vibrio cholerae were negative.The detection limitation was 19 cfu/μl,while for simulated samples was 260 cfu/μl.The t HDA assay was used to detect 18 actual samples,9 of them were positive for Salmonella,which was consistent with the result of national standard method.Conclusion The t HDA assay for Salmonella was rapid,sensitive and specific,and will play an important role in the detection and surveillance of food-borne diseases.