检测气单胞菌属的SYBR Green荧光PCR方法的建立和评价  

Establishment and evaluation of SYBR Green fluorescent PCR for detection of Aeromonas

作　　者：杜小莉[1] 刘九银 洪颖[2] 王利[2] 江良梁[2] 陈谨[2] 卢昕[1] 阚飙[1] 周海健[1] 崔志刚[1] Du Xiaoli;Liu Jiuyin;HongYing;Wang Li;Jiang Liangliang;Chen Jin;Lu Xin;Kan Biao;Zhou Haijian;Cui Zhigang(State Key Laboratory for Infectious Disease Prevention and Control,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Maanshan Prefectural Center for Disease Control and Prevention,Maanshan 243000,Anhui,China)

机构地区：[1]中国疾病预防控制中心传染病预防控制所,传染病预防控制国家重点实验室,北京102206 [2]马鞍山市疾病预防控制中心,安徽马鞍山243000

出　　处：《疾病监测》2020年第5期425-429,共5页Disease Surveillance

基　　金：国家科技重大专项(No.2018ZX10714002-004)。

摘　　要：目的建立一种检测气单胞菌的SYBR Green荧光PCR方法。方法使用文献报道的气单胞菌属16S rRNA基因扩增引物,建立SYBR Green荧光PCR检测方法,评价其特异性、灵敏度和实际应用价值。结果SYBR Green荧光PCR方法的检测下限为10^-7 ng/ml,检测的21株气单胞菌均为阳性,检测的弧菌、大肠埃希菌、沙门菌、小肠结肠炎耶尔森菌、志贺菌均为阴性。针对104份健康人群粪便样本,8.65%的样本分离到气单胞菌,28.85%的样本SYBR Green荧光PCR方法检测阳性。其中8份标本采用2种方法检测均为阳性。样本核酸检测阳性后再进行病原菌的分离,会漏检1份样本(11.11%),但可以减少71.15%(74/104)的分离培养及鉴定工作。结论SYBR Green荧光PCR方法具有很好的特异性和敏感度,用于大样本的早期筛查可以减少70%左右的工作量,适合应用于气单胞菌感染和携带的监测。Objective To establish a SYBR Green fluorescent PCR assay for the detection of Aeromonas.Methods SYBR Green fluorescent PCR was established by using 16 S rRNA gene amplification primers of Aeromonas reported,and its specificity,sensitivity and practical application value were evaluated.Results The detection limit of SYBR Green fluorescent PCR was 10–7 ng/ml.The assay to detect 21 Aeromonas strains showed positive results,but the assay showed negative results for Vibrio,Escherichia coli,Salmonella,Yersinia enterocolitica and Shigella.For 104 stool samples of healthy people,Aeromonas was isolated from 8.65% of the samples,28.85% of the samples showed positive results in SYBR Green fluorescent PCR.Eight of them were positive by both methods.If the pathogen was isolated after positive SYBR Green fluorescent PCR detection,one sample(11.11%)would be missed,but 71.15%(74/104)of the workload of isolation,culture and identification might be reduced.Conclusion SYBR Green fluorescent PCR has high specificity and sensitivity.It can reduce the workload by about 70% in the early screening of large samples,and is suitable for the monitoring of Aeromonas infection and carrier.

关 键 词：气单胞菌 荧光PCR 分子检测 

分 类 号：R378[医药卫生—病原生物学]