摘要
目的建立以invA特异性基因设计合成为引物的沙门氏菌环介导等温扩增(LAMP)快速检测方法。方法选择沙门氏菌的毒力因子invA基因作为检测模板,采用Primer Exploer V5软件设计3组LAMP引物[正向外引物(F3)、反向外引物(B3),正向内引物(FIP)、反向内引物(BIP),正向环引物(LF)、反向环引物(LB)],以4种沙门氏菌(甲型副伤寒沙门氏菌、乙型副伤寒沙门氏菌、丙型副伤寒沙门氏菌、鼠伤寒沙门氏菌)和3种非沙门氏菌(金黄色葡萄球菌、大肠杆菌、产志贺氏毒素大肠埃希氏菌)的细菌DNA为模板,进行LAMP快速检测。结果在反应温度为65℃、反应时间为60 min条件下,即可快速检测4个亚种沙门氏菌,其检测灵敏度可达10 cfu/mL,且试验结果易于观察,筛选的引物特异性良好。结论所建立的方法灵敏度高、特异性好、检测快速,可应用于中成药中沙门氏菌的快速检测。
Objective To establish a rapid detection method of loop-mediated isothermal amplification(LAMP)for Salmonella using invA specific gene to design and synthetize primers.MethodsThe virulence factor invA gene of Salmonella was used as the detection template.Three groups of LAMP primers[forward outer primer(F3)and backward outer primer(B3),forward inner primer(FIP)and backward inner primer(BIP),forward loop primer(LF)and backward loop primer(LB)]were designed by the Primer Exploer V5 software.The DNA of four Salmonella species(Salmonella paratyphi A,Salmonella paratyphi B,Salmonella paratyphi C and Salmonella typhimurium)and three non-Salmonella species[Staphylococcus aureus,Escherichia coli and Shiga toxin-producing Escherichia coli(STEC)]were used as the templates for the LAMP rapid detection.ResultsFour Salmonella subspecies could be rapidly detected under the conditions of reaction temperature of 65℃and reaction time of 60 min.The sensitivity of detection could reach 10 cfu/mL,the results of detection were easy to observe,and the screening primers were specific.ConclusionThe established method is sensitive,specific and rapid,which can be used for the rapid detection of Salmonella in Chinese patent medicine.
作者
冯润东
贾首前
王莉芳
杨晓莉
徐长根
张鸿豪
FENG Rundong;JIA Shouqian;WANG Lifang;YANG Xiaoli;XU Changgen;ZHANG Honghao(Shaanxi Institute for Food and Drug Control,Xi'an,Shaanxi,China 710065;National Medical Products Administration Key Laboratory of Drug Microbiological Testing Technology,Xi'an,Shaanxi,China 710065;Shaanxi Medical Products Administration,Xi'an,Shaanxi,China 710065)
出处
《中国药业》
CAS
2023年第5期83-87,共5页
China Pharmaceuticals
基金
陕西省重点研发计划项目[2019SF-011]。
作者简介
第一作者:冯润东,男,硕士研究生,副主任药师,研究方向为药理毒理及安全性评价,(电子信箱)43474735@qq.com;通信作者:徐长根,女,硕士研究生,主任药师,研究方向为药品食品分析、药理毒理及安全性评价,(电子信箱)345154532@qq.com。
