摘要
目的:建立一种快速、特异、敏感的分子生物学诊断方法对腹泻患者粪中痢疾杆菌致病基因ipaH及ial进行检测.方法:分别应用常规培养生化血清学鉴定方法、普通PCR、巢氏PCR(nested-PCR)方法,对71例腹泻患者粪标本进行检测.痢疾杆菌致病基因ipaH及ial的扩增产物经电泳分离.结果:在71例腹泻患者粪标本中,常规培养生化血清学鉴定方法分离出福氏痢疾杆菌24例,宋内痢疾杆菌23例,志贺痢疾杆菌1例,沙门菌23例.采用普通PCR法检测痢疾杆菌粪标本48例,ipaH基因均阳性(100%);ial基因阳性34例(70.8%),余14例为阴性.对该14例粪标本进而采用巢氏PCR法进行ial基因扩增,其中12份标本阳性.48例痢疾杆菌粪标本中46例ipaH和ial基因为双阳性(46/48).沙门菌粪标本23例中ipaH及ial基因表达均为阴性.以上两种方法的诊断一致性检验Kappa=0.937,差异有统计学意义(P<0.05).71例腹泻患者粪标本采用PCR和巢氏PCR法扩增得到的ipaH和ial基因特异片段与基因库中发表的标准菌株序列比较,一致性为100%.结论:建立了快速诊断腹泻患者粪中痢疾杆菌致病基因ipaH和ial的PCR检测方法,具有简便、快速、特异和敏感的特点.
AIM： To establish a diagnostic method fast, specific and sensitive for detecting the invasion plasmid antigen H （ipaH） and the invasionassociated locus （ial） DNA sequence of Shigella.
METHODS： Seventy-one stool specimens from patients with acute diarrhea were tested by two methods; a traditional culture method, and a polymerase chain reaction （PCR） and nested-PCR assay. PCR-amplified products were initially evaluated by electrophoresis with 20 g/L agarose gel and visualization by ethidium bromide staining.
RESULTS： Serological assay classified the Shigella isolates as follows： 24 Shigella flexneri, 23 Shigella sonnei and one Shigella dysenteriae. Salmonella was isolated from 23 of the 71 stool specimens by conventional coproculture. PCR assays showed that 46 of the 48 Shigella spp. were positive for the ipaH gene, 34 were positive for the ial gene, and 14 were negative. Nested-PCR assays detected 14 specimens as negative for ial, 12 were positive, and 46 specimens were positive for both the ipaH and ial genes. The ipaH and ial genes were both negative in 23 Salmonella isolates. The kappa test, was used to compare two test results, and the kappa value was 0.937 （P 〈 0.05）.
CONCLUSION： PCR assays detecting the invasion-associated genes of Shigella from feces provided fast, specific and sensitive diagnostic methods.
出处
《世界华人消化杂志》
CAS
北大核心
2007年第19期2128-2132,共5页
World Chinese Journal of Digestology
