酶促重组酶扩增-侧流层析快速检测沙门菌的方法研究

Rapid detection of Salmonella by enzymatic recombinase amplification combined with lateral flow chromatography

目的建立一种酶促重组酶扩增(ERA)和侧流层析技术(LF)相结合的沙门菌快速检测方法,为沙门菌现场检测提供技术支持。方法根据沙门菌属高度保守的鞭毛基因fimY设计特异性ERA引物,采用毛细管电泳筛选合适的引物,再设计探针,对扩增温度和时间进行优化,建立ERA-LF检测体系对菌株DNA进行扩增,采用侧向流试纸条检测产物。采用鼠伤寒沙门菌标准菌株检验方法的灵敏度,采用10种其他肠道感染标准菌株检验特异度,采用实际分离到的沙门菌验证对实际样本检测能力。结果经过毛细管电泳筛选获得沙门菌特异性引物,在扩增温度为37℃、反应时间为20 min的条件下,最低检测浓度为5 copies/μL。本方法对沙门菌核酸扩增结果为阳性,10株其他肠道感染菌核酸扩增结果均为阴性,特异度较好。对100株(包含10种血清型)沙门菌进行检测,结果均显示阳性。结论本研究建立的ERA-LF方法可用于沙门菌的现场即时检测。

Objective To establish a rapid detection method for Salmonella based on the combination of enzymatic re-combinase amplification(ERA)and lateral flow chromatography(LF),so as to provide technical support for the on-site detection of Salmonella.Methods Specific ERA primers and probes were designed based on the highly conserved fla-gella gene fimY in Salmonella.The primers were screened using capillary electrophoresis,and the probes were designed according to the amplification range of the screened primers.The amplification temperature and time were optimized to establish the amplification method,and the product was detected using LF strips.A standard strain of Salmonella was used to verify the sensitivity,10 other gut bacteria were used to to verify the specificity and sensitivity,and the nucle-ic acid of the actual Salmonella strains was amplified to verify the detectability.Results After screening for Salmonel-la-specific primers using capillary electrophoresis,the minimum detection concentration was 5 copies/μL under the am-plification temperature of 37℃and reaction time of 20 minutes.This method had a positive amplification result for Salmonella nucleic acid,and the amplification results of 10 other gut bacteria were all negative,with good specificity.Conclusion This method provides a possibility for on-site point of care testing of Salmonella infection.