摘要
该研究拟采用沙门氏菌核酸标准物质验证实时荧光定量PCR检测沙门氏菌,并对沙门氏菌核酸标准物质的适用性、均匀性、稳定性进行考察。使用不同的PCR仪进行检测比较,建立不同的PCR酶体系,并在沙门氏菌核酸标准物质中添加保护剂,以此来验证沙门氏菌核酸标准物质的适用性;同时根据线性图、参数表对沙门氏菌核酸标准物质进行均匀性、短期稳定性和反复冻融稳定性分析。结果表明研究设计的荧光定量PCR方法辅以标准物质具有良好的种属特异性,适用于常见的PCR仪和不同的酶体系,保护剂不影响沙门氏菌核酸的荧光扩增。研制的沙门氏菌标准物质样品均匀,未稀释的沙门氏菌核酸检测样在实验中6 d内量值保持稳定,反复冻融10次后的量值也相对稳定,溶解于保护剂的低浓度沙门氏菌核酸检测样稳定性加强,但低浓度沙门氏菌核酸检测样短期稳定性及反复冻融实验量值不稳定。辅以标准物质的实时荧光定量PCR技术检测准确度高且稳定,可用来验证各实验室的检测能力,实现微生物核酸检测技术的快速、准确、可溯源。
Salmonella is a kind of food-borne pathogen which belongs to Enterobacteriaceae.However,the content of Salmonella in food is usually low,the routine test is time-consuming,so the detection with high sensitivity,repeatability and accuracy is needed.The nucleic acid reference material of Salmonella was used to verify the real-time fluorescence quantitative PCR(qRT-PCR)method for detecting Salmonella,the applicability,uniformity and stability of nucleic acid reference material of Salmonella were investigated in this study.To optimize the nucleic acid reference materials for verifying qRT-PCR method in detection of Salmonella,various enzyme systems for PCR and protective agents for nucleic acid reference materials were studied.The homogeneity,short-term stability and repeated freeze-thaw stability of nucleic acid reference materials were analyzed based on the linear graph and parameter table.The results showed that qRT-PCR method with relevant nucleic acid reference materials in this study was specific to Salmonella amplification,it was applicable to commonly used PCR instruments and various enzyme systems.The protective agent in reference materials did not inhibit the fluorescence amplification of Salmonella.The quality evaluation of reference material indicated that Salmonella nucleic acid samples were uniform,it remained stable within 6 days in the stability test without dilution.The quantity was stable after repeated freezing and thawing for 10 times.And the stability of diluted Salmonella nucleic acid sample was enhanced after adding protective agent.However,the short-term stability and quantity of diluted Salmonella nucleic acid samples were unstable in repeated freezing and thawing tests.The RT-PCR with reference material has high accuracy and stability in detecting Salmonella.It can be used to verify the detection ability of laboratories in rapidly and accurately detecting microbial nucleic acid with traceability.
作者
王菲
张玲
杨佳怡
叶子弘
俞晓平
WANG Fei;ZHANG Ling;YANG Jiayi;YE Zihong;YU Xiaoping(College of Life Sciences,China Jiliang University,Hangzhou 310018,China;National Institute of Metrology,Beijing 100013,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2020年第8期219-225,共7页
Food and Fermentation Industries
基金
国家重点研发计划项目(2018YFC1200502,2018YFE0201602,2017YFF0204600,2017YFF004601)。
作者简介
第一作者:王菲,硕士研究生;通讯作者:俞晓平,研究员,E-mail:yxp@cjlu.edu.cn。
