酿脓链球菌特异、快速分子检测方法的建立  

Specific and rapid molecular detection method for Streptococcus pyogenes

作　　者：王玉贤 王莉芝 唐雪 赵怡环 李秉成 黄维藻[3] 雍彬[2] 陶向 WANG Yuxian;WANG Lizhi;TANG Xue;ZHAO Yihuan;LI Bingcheng;HUANG Weizao;YONG Bin;TAO Xiang(Chengdu Institute of Product Quality Inspection Co.,Ltd.,Chengdu 610199,China;College of Life Sciences,Sichuan Normal University,Chengdu 610101,China;Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,China)

机构地区：[1]成都产品质量检验研究院有限责任公司,四川成都610199 [2]四川师范大学生命科学学院,四川成都610101 [3]中国科学院成都生物研究所,四川成都610041

出　　处：《中国测试》2023年第5期74-81,共8页China Measurement & Test

基　　金：四川省市场监督管理局科技计划项目(SCSJ2020012)。

摘　　要：集中空调内部容易滋生细菌、真菌等微生物,成为有害微生物污染传播和扩散的媒介。为对公共场所空调系统有害微生物酿脓链球菌(Streptococcus pyogenes)进行有效检测与预防,该研究建立该菌的高效、特异的PCR检测方法和重组酶等温扩增(recombinase aided amplification,RAA)结合侧流层析试纸条(lateral flow dipstick,LFD)的快速恒温检测方法。基于序列的相似性比对方法,从酿脓链球菌基因组序列中挖掘到5个特异的DNA区段,长度分别为5348、1098、5287、5369、240 bp。以5369 bp的特异DNA片段为检测靶标,设计PCR引物,优化确定最优退火温度为60.0℃;对PCR引物的特异性进行分析,确定该引物能有效区分酿脓链球菌的多个近缘种;当PCR循环数为25、30、35时,最低可检出400 fg/μL(1.248×105拷贝/μL)、4 fg/μL(1.248×103拷贝/μL)、0.4 fg/μL(1.248×102拷贝/μL)的模板DNA。设计RAA-LFD引物及探针,优化反应体系,确定最优反应温度为37℃;对RAA-LFD引物及探针进行特异性分析,确定该引物能有效区分酿脓链球菌的多个近缘种;当反应时间为5、10、15、20、25 min时,最低可检出400 fg/μL(1.248×105拷贝/μL)、4 fg/μL(1.248×103拷贝/μL)、4×10–1 fg/μL(1.248×102拷贝/μL)、4×10–2 fg/μL(1.248×101拷贝/μL)、4×10–5 fg/μL(1.248×10–2拷贝/μL)。建立的酿脓链球菌的PCR检测体系和RAA-LFD快速检测体系特异性强、灵敏度高、快速高效,为公共场所集中空调系统有害微生物酿脓链球菌的检测、防控提供新的技术选择。It is easy for bacteria,fungi and other microorganisms to grow inside the centralized air conditioner,and this can make centralized air conditioner become easily the medium for the spread of harmful microorganisms.In order to effectively detect and prevent Streptococcus pyogenes in air conditioning systems in public places,this study developed an efficient and specific PCR detection and an recombinase aided amplification-lateral flow dipstick(RAA-LFD)system for S.pyogenes.Based on the sequence similarity alignment,five species specific genomic DNA fragments were identified from the genome sequence of S.pyogenes with lengths of 5348,1098,5287,5369 and 240 bp,respectively.The specific DNA fragment of 5369 bp was used as detection target,PCR primers were designed.The optimal PCR annealing temperature was 60.0℃,and the primers could effectively distinguish multiple closely related species of S.pyogenes.When the number of PCR cycles was 25,30 and 35,the DNA detection sensitivity were 400 fg/μL(1.248×105 copies/μL),4 fg/μL(1.248×103 copies/μL)and 0.4 fg/μL(1.248×102 copies/μL),respectively.RAA-LFD primers and probes were also designed.The optimal reaction temperature of RAA was 37°C.it can also effectively distinguish multiple related species of S.pyogenes.When the reaction time is 5,10,15,20,and 25 min,the DNA detection sensitivity of RAA-LFD were 400 fg/μL(1.248×105 copies/μL),4 fg/μL(1.248×103 copies/μL),4×10-1 fg/μL(1.248×102 copies/μL),4×10-2 fg/μL(1.248×101 copies/μL),and 4×10-5 fg/μL(1.248×10-2 copies/μL),respectively.The PCR detection system and RAA-LFD rapid detection system for S.pyogenes developed in this study have high specificity,sensitivity,rapidity and efficiency,providing new technical options for the detection,prevention and control of S.pyogenes.

关 键 词：酿脓链球菌 物种特异 基因组DNA PCR检测 RAA-LFD快速检测 

分 类 号：R126.4[医药卫生—环境卫生学] TB9[医药卫生—公共卫生与预防医学]