利用实时荧光定量-聚合酶链反应方法检测粪便标本中的伤寒沙门菌

Detection of Salmonella typhi in stool samples with real-time fluorescent quantitative polymerase chain reaction

目的建立针对粪便标本中伤寒沙门菌的检测方法,评价该方法的特异性、敏感性及检测下限,以提高感染人群粪便标本中伤寒沙门菌的检出率。方法根据伤寒沙门菌特异基因STY1633设计特异性引物,优化反应条件,建立实时荧光定量-聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,qPCR)反应体系。利用92株常见的非伤寒病原菌及44株伤寒沙门菌的染色体DNA评价该方法的特异性和灵敏性,并对伤寒沙门菌的粪便模拟标本进行检测下限评价,同时利用10份伤寒患者粪便标本及48份其他病原所致发热和/或伴腹泻的患者粪便标本进行特异性、敏感性验证。结果利用本方法检测的伤寒沙门菌纯菌及伤寒患者标本均扩增阳性,其余的非伤寒沙门菌、致腹泻的其他5种肠道致病菌及引起发热症状的8种常见非肠道病原菌纯菌及相应患者标本均扩增阴性。在对纯伤寒沙门菌DNA检测中,qPCR法的最低检测限为500 fg/反应,相当于97个拷贝/反应。以粪便模拟样品提取DNA为模板的检测中,增菌前检测下限达104cfu/g,增菌后可达50 cfu/g。结论基于STY1633基因的实时荧光定量PCR方法在检测粪便中的伤寒沙门菌中具有很好的特异性、灵敏度,为伤寒沙门菌的快速诊断及某些不明原因发热及腹泻症状的病原初步鉴定提供了新的简便型手段,对于伤寒的早期诊断及预防控制提供了技术支持。

Objective To establish a real-time fluorescent quantitative polymerase chain reaction(qPCR) assay to detect Salmonella typhi in stool samples.Methods Primers were designed based on the sequence of STY1633,specific gene of S.typhi,for qPCR assay.The specificity and sensitivity of the qPCR assay established were evaluated and verified by detecting 92 strains of common pathogens other than S.typhi and 44 S.typhi strains.The detection limit was determined by using simulative stool specimen supplemented with S.typhi.Further verifying of the specificity and sensitivity of the qPCR assay was performed with clinical stool samples from patients with fever and /or diarrheal symptoms.Results Total 44 S.typhi isolates were amplified to be positive by this qPCR assay.Five enteric pathogens other than S.typhi causing diarrhea and 8 enteric non-enteric bacteria causing bacteremia with fever symptom were detected to be negative.For purified total DNA from pure culture isolates,the detection limit of the qPCR assay was 500 fg per reaction,equal to 97 copies per reaction.The limit of detection was 104 cfu/g and 50 cfu/g respectively with crude nucleotide from simulative stool samples before and after enrichment.Conclusion The qPCR assay for detecting S.typhi in stool sample with high sensitivity and specificity was established,which could be used in the rapid diagnosis of S.typhi infection and differential diagnosis of other pathogen infections with fever and diarrheal symptoms as well as in the early warning,prevention and control for typhoid fever.