口腔幽门螺杆菌基因定量测定方法的建立及其应用评价  

Establishment and application evaluation of quantitative detection method of Helicobacter pylori in oral cavity

作　　者：别明江[1] 严谨[2] 王保宁[1] 谢艳[2] 祝捷 王馨田 王红仁[1] 李明远[1] 杨靖[5] 李尤玲[6] 

机构地区：[1]四川大学华西基础医学与法医学院,四川成都610041 [2]四川大学华西医院消化内科,四川成都610041 [3]四川万可泰生物技术有限责任公司,四川成都610041 [4]成都树德中学,四川成都610031 [5]湖北医药学院附属人民医院感染性疾病科,湖北十堰442000 [6]十堰巿人民医院中西结合科,湖北十堰442000

出　　处：《西部医学》2017年第1期17-21,共5页Medical Journal of West China

基　　金：四川省科技厅应用基础研究项目(2014JY0201);湖北医药学院研究生启动金资助计划项目(2015QDJZR08);十堰市科技局研究与开发专项基金项目(16K70);成都市科技局重点科研项目(2014-CP02-00079-GX)

摘　　要：目的建立一种通过口腔唾液的特异、灵敏测定幽门螺杆菌核酸DNA的荧光定量PCR方法,并对其进行临床比对评价,为快速、准确、无创检测幽门螺杆菌感染提供新方法。方法以高度保守的幽门螺杆菌16srRNA序列为靶序列设计引物,经Blast软件对相似序列比对后,筛选出一对最优并与常见胃肠道细菌无交叉反应的引物;用荧光定量PCR扩增,对引物的退火温度及反应体系进行优化,确立反应条件;用已知菌落数(CFU/ml)的幽门螺杆菌菌悬液与唾液混合,梯度稀释扩增测试后进行灵敏度分析,建立幽门螺杆菌菌落数(CFU/ml)和荧光定量PCR循环数(Ct)关系的标准曲线;用乳酸杆菌、金黄色葡萄球菌、奈瑟氏菌、小齿类杆菌和唾液链球菌的基因进行特异性分析;利用此方法对150例来自临床胃镜中心的已完成尿素酶(URT)测定,并自愿参加研究者的口腔唾液标本进行收集检测,结果与同一病人的14 C呼气实验检测结果进行比对分析,计算阳性率、阴性率和符合率。结果本检测方法的引物退火温度是62℃,反应体系10μl;对幽门螺杆菌的检测敏感度为102 CFU/ml;本检查与乳酸杆菌、金黄色葡萄球菌、奈瑟氏菌、小齿类杆菌和唾液链球菌无交叉反应;本方法与14 C呼气实验检测结果比较灵敏度74.12%,特异度86.15%,符合率79.33%,Kappa值为0.59。结论建立了口腔唾液幽门螺杆菌基因检测的分子诊断方法;该方法的特异性好,灵敏度高,为临床检测口腔幽门螺杆菌提供了一个无创、方便的新方法。Objective To establish a simple, specific, sensitive PCR method with oral saliva sample to detect Helicobacter pylori and evaluated the sensitivity and specificity for detection of the helicobacter pylori infection in the clinical field. Methods Primers and probes were designed by corresponding to the converse 16s rRNA gene sequences and which were matched by Blast method in Genebank and then. The excellent primers and probes were selected. Electrophoretic analysis of the PCR products was amplified by different annealing temperature and different reaction system. The best reaction conditions were determined. The counted Hicobacter pylori in bacterium suspension and oral saliva mixture were serially diluted. The gene was extracted, and amplificated by real time PCR. The sensation was evaluated, and the standard curve of the colony form unit （CFU） counts corresponding to the real time PCR cycle numbers were established. The genes of pathogens, such as Lactohacilli strain, Saphylococcusaureus, Neisseriaceae, B. denticola and S. salivarius were used for specificity test. The saliva samples of 150 patients were detected by real time PCR and compared the results with the URT. Results The primer annealing temperature at 62℃ and reaction volume was 10μl. Its sensitivity was able to detect 102 CFU/ml of Hicobacter pylori and no cross reaction with Lactobacilli strain, Saphylococcusaureus, Neisseriaceae, B. denticola and S. salivarius. The sensitivity compared with URT was 74. 12%, specificity was 86. 15%, the coincidence rate was 79.33% and Kappa was 0.59, respectively. Conclusion A new gene saliva diagnosis method to Helicobacter pylori was established. The limit of detection（LOD） to Hicobacter pylori was 102 CFU/ml and no cross reaction with other pathogens. The sample was saliva which could give the doctor a new sensitive, specific, rapid, noninvasive and available diagnostic tool to detect Helicobacter pylori in oral cavity.

关 键 词：唾液 幽门螺杆菌 基因 定量检测 

分 类 号：R573.6[医药卫生—消化系统]