多重PCR检测唾液标本中的幽门螺杆菌16S rRNA及cagA基因  

Combined detection of Helicobacter pylori 16S rRNA and cagA gene in saliva specimens using multiplex PCR

作　　者：王鑫莹 孙丽媛 杨志平[2] 宋顺佳 李南 刘悦 田婉佳 赵云冬 WANG Xinying;SUN Liyuan;YANG Zhiping;SONG Shunjia;LI Nan;LIU Yue;TIAN Wanjia;ZHAO Yundong;无(College of Medical Technology,Beihua University,Jilin 132013,China;Department of Gastroenterology of Beihua University Affiliated Hospital,Beihua University,Jilin 132013,China;Reproductive Medicine Prenatal Genetics Center,Bethune First Hospital of Jilin University,Changchun 130021,China)

机构地区：[1]北华大学医学技术学院,吉林吉林132013 [2]北华大学附属医院消化内科,吉林吉林132013 [3]吉林大学白求恩第一医院生殖中心-产前诊断中心,吉林长春130021

出　　处：《南方医科大学学报》2021年第12期1816-1821,共6页Journal of Southern Medical University

基　　金：吉林省教育厅“十三五”科学技术项目(JJKH20190657KJ,JJKH20180365KJ);吉林省科技发展计划项目(20190304115YY);北华大学研究生创新计划项目(北华研创合字[2019]026,北华研创合字[2019]015,北华研创合字[2019]069)。

摘　　要：目的检测唾液标本幽门螺杆菌(Hp)的16S rRNA基因及cagA基因,了解消化道疾病患者口腔中Hp存在情况及致病情况。方法利用生物学信息技术,设计Hp 16S rRNA、cagA基因特异性引物,建立检测Hp 16S rRNA、cagA基因的多重PCR方法;重组克隆质粒构建标准阳性对照品;收集156例消化道疾病患者的唾液标本,提取唾液标本中细菌DNA,应用建立的多重PCR方法,检测Hp 16S rRNA基因及cagA基因,并对结果进行分析。结果建立的检测Hp 16S rRNA基因及cagA基因的多重PCR方法特异性强,灵敏性较高,最低检测线为10^(3) copies·μL^(-1);重组质粒pGMT-16s和pGMT-cagA,可作为鉴定Hp的标准阳性对照品;检测Hp感染的消化道疾病患者的唾液标本,口腔携带Hp 16S rRNA基因的阳性率为87.2%,cagA基因阳性率为23.1%。结论Hp感染的消化道疾病患者口腔唾液标本中存在Hp,口腔中存在Hp可能是诱发消化道Hp感染及治疗后复发的一个重要原因。Objective To establish a multiplex PCR-based method for detecting Helicobacter pylori(Hp)16S rRNA gene and cagA gene in saliva samples for investigating the prevalence of Hp in the oral cavity of Hp-infected patients with digestive tract diseases.Methods Bioinformatics technique was used to design specific primers for Hp 16S rRNA and cagA genes for Hp detection using multiplex PCR,with recombinant cloning plasmids serving as the standard positive control.Oral saliva samples were collected from 156 patients with digestive tract diseases,and Hp 16S rRNA and cagA genes were detected using the established multiplex PCR system.Results The established multiplex PCR system showed a strong specificity and a high sensitivity for detecting Hp 16S rRNA gene and cagA gene,with the lowest detection limit of 103 copies/μL.The recombinant plasmids pGMT-16s and pGMT-cagA could be used as standard positive controls for the identification of Hp.Among the 156 saliva samples,87.2% were positive for Hp 16S rRNA gene and 23.1% for Hp cagA gene.Conclusion Hp is highly prevalent in saliva specimens of Hp-infected patients with digestive tract diseases.The presence of Hp in the oral cavity may importantly contribute to Hp infection in the digestive tract and recurrence after treatment.

关 键 词：幽门螺杆菌 16S rRNA基因 CAGA基因 唾液 

分 类 号：R573[医药卫生—消化系统] R440[医药卫生—内科学]