一种检测肝螺杆菌套式PCR方法的建立及临床应用  

Development and clinical application of a nested-PCR assay for Helicobacter hepaticus detection

作　　者：秦和平[1] 毕丽红[2] 韦碧柳[1] 潘信义[1] 叶安丽[1] 易平[3] 

机构地区：[1]柳州市人民医院医疗保健部,广西壮族自治区柳州市545006 [2]南方医科大学附属南方医院消化内科,广东省广州市510515 [3]柳州市人民医院消化内科,广西壮族自治区柳州市545006

出　　处：《世界华人消化杂志》2015年第28期4507-4514,共8页World Chinese Journal of Digestology

基　　金：广西省卫生厅基金资助项目;No.Z2013651~~

摘　　要：目的:建立一种敏感性高、特异性强的检测肝螺杆菌(Helicobacter hepaticus,H.hepaticus)的套式PCR方法并对其临床应用进行探讨.方法:本研究基于9种H.hepaticus亚种的16S rRNA基因设计筛选出1套套式引物(2条内引物、2条外引物),优化反应条件后,通过模拟粪便和胆汁标本、相关细菌、动物模型标本和临床标本的检测对该套式PCR方法的敏感性、特异性进行评估.结果:在模拟粪便和胆汁标本中,该方法检测H.hepaticus的最低检出浓度达到102C F U/m L.幽门螺杆菌、胆螺杆菌、空肠弯曲菌、大肠埃希菌、粪肠球菌、铜绿假单胞菌、金黄色葡萄球菌检测结果均为阴性.本研究建立了3例感染肝螺杆菌的SPF级Balb/c小鼠模型.该方法在3例模型的粪便和盲肠标本及2例模型的肝脏标本中检测出H.hepaticus.10例胆石病患者中,该法在2例患者的胆囊胆汁、胆囊黏膜和粪便标本中检测出有H.hepaticus临床标本的检测中,PCR检测方法、医疗干预和标本的类型及处理可能都会影响检测的结果.结论:本研究建立的套式P C R方法,敏感性高、特异性强,可以用于小鼠和人的H.hepaticus感染.H.hepaticus感染可能和人胆石病有关.PCR检测方法、医疗干预和标本的类型及处理可能都会影响临床标本的检测结果.AIM：To develop a nested-PCR assay for the detection of Helicobacter hepaticus（H.hepaticus）with a high sensitivity and specificity and to analyze its clinical application.METHODS：The nested primers were designed based on the sequences of 16 S rRNA gene of nine subtypes of H.hepaticus.After optimizing reaction condition,the sensitivity and specificity of the assay were examined using simulated feces and bile samples,related bacteria,mouse infection model and clinical inpatients samples.RESULTS：The detection limit for H.hepaticus strain in simulated feces and bile samples was10~2 CFU/mL.No specific PCR product was detected with DNA from Helicobacter pylori,Helicobacter bilis,Campy lobacter jejuni,Escherichia coli,Enterococcus faecalis,Pseudomonas aeruginosa and Staphylococcus aureus.H.hepaticus was successfully detected in the liver,caecum and feces of experimentally infected mice.Moreover,H.hepaticus was successfully detected in the gallbladder bile,gallbladder mucosa and fecal samples from two of ten patients with cholelithiasis.PCR methods,medical intervention,sample type and management may all influence the detection.CONCLUSION：Due to the high sensitivity and specificity of the PCR assay,it may be used to detect H.hepaticus infection.H.hepaticus may be associated with the pathogenesis of human cholelithiasis.PCR methods,medical intervention,sample type and management may all influence the detection of clinical samples.

关 键 词：肝螺杆菌 套式PCR 16S RRNA 临床应用 

分 类 号：R440[医药卫生—诊断学]