摘要
目的建立实时荧光定量反转录-聚合酶链反应(real time fluorescent quantitative reverse transcription-polymerase chain reaction,rRT-PCR)方法检测伤寒沙门菌。方法针对伤寒沙门菌STY1631基因设计特异性引物,通过优化反应条件,建立检测该靶基因的rRT-PCR方法,利用51个血清型的纯培养伤寒和非伤寒沙门菌菌株、常见非沙门致腹泻病原菌以及发热为主要症状的8种常见病原菌核糖核酸(RNA)评价该方法的特异性及敏感性,同时对伤寒沙门菌全血模拟标本进行敏感性检测。结果利用该方法检测48株伤寒沙门菌均阳性,其余33种非伤寒沙门菌血清型、致腹泻的其他5种肠道致病菌以及发热为主要症状的8种常见非沙门菌也均扩增阴性。在对纯菌总RNA检测中,rRT-PCR的最低检测限为1 pg/反应,即194个拷贝/反应。以全血模拟样品提取核酸为模板的检测中,敏感性达1×102cfu/ml。结论建立了敏感性高、特异性好的rRT-PCR检测血液中伤寒沙门菌的方法,为伤寒沙门菌感染的快速诊断及不明原因发热症状的病原初步鉴别提供了简便的手段,可用于对伤寒的早期诊断、预防控制及临床治疗。
Objective To establish an assay of real time fluorescent quantitative reverse transcriptive polymerase chain reaction （rRT-PCR） to detect Salmonella Typhi in blood. Methods Specific primers STY1631 gene of S. Typhi were designed and modified to establish rRT-PCR assay and the specificity and sensitivity of rRT-PCR with RNA as template were evaluated and verified by checking cultured Salmonella spp. in 51 serotypes, predominant non-salmonella enteric diarrheal pathogens and eight species of bacteria inducing bacteremia with fever as major symptom. The simulative blood specimens supplemented with S. Typhi were tested by the rRT-PCR assay. Results The established rRT-PCR assay successfully detected STY1631 gene of S. Typhi. Totally 48 S. Typhi isolates were amplified to be positive. Other isolates, including non-salmonella strains in 33 serotypes, predominant non-salmonella enteric diarrheal pathogens and eight species of bacteria causing bacteremia with fever, were amplified to be negative. For purified total RNA from pure cultured isolates, the detection limit of the assay was 1 pg per reaction, equal to 194 copies per reaction. The sensitivity achieved 1 x 102 cfu /ml with the purified nucleotide from simulative blood. Conclusion The rRT-PCR assay for detecting S. Typhi with high sensitivity and specificity was established, which would be suitable for the rapid diagnosis of S. Typhi infection and identification of other pathogens causing fever for the early warning, prevention and treatment of typhoid fever.
出处
《疾病监测》
CAS
2012年第6期471-474,共4页
Disease Surveillance
基金
国家十二五重大专项-重大传染病应急处置检测技术平台(No.2011ZX10004-001)~~
