摘要
目的建立反转录-环介导等温核酸扩增（RT-LAMP）方法特异检测甲型副伤寒沙门菌。方法针对甲型副伤寒沙门菌特异保守hsdM基因设计引物,通过优化反应条件,建立检测该靶基因的RT-LAMP体系。利用多种血清型沙门菌及非沙门菌菌株DNA评价该方法的特异性及敏感性。同时对甲型副伤寒沙门菌全血模拟标本及粪便模拟标本进行敏感性检测,并利用临床样本进行初步验证。结果等温65℃条件下,30～60 min内完成RTLAMP反应,利用该方法检测130株甲型副伤寒沙门菌均为阳性,其他非甲型副伤寒沙门菌均扩增阴性。对纯菌RNA检测中检测下限为50 pg/反应,约为19 000个拷贝/反应。在以全血模拟样本的检测中,敏感性达80 cfu/ml,略高于PCR检测低限。对粪便模拟标本的检测中,对原始样品检测下限为500 cfu/g;增菌后样品检测下限为0.8 cfu/g,比PCR检测低限高2～10倍。临床样本中甲型副伤寒患者血液标本检测均阳性,而伤寒样本检测均阴性。结论建立了敏感性高、特异性好的检测甲型副伤寒沙门菌的RT-LAMP方法,为甲型副伤寒沙门菌感染的快速诊断提供了简易手段,可试用于甲型副伤寒的早期诊断。
Objective To establish a rapid assay by reverse transcription loop-mediated isothermal amplification（ RTLAMP） to detect Salmonella paratyphi A. Methods The specific primers recognizing distinct regions of hsdM gene of S. paratyphi A were designed and modified to establish RT-LAMP assay and the specificity and sensitivity of RT-LAMP with RNA as template were evaluated and verified by detecting cultured Salmonella and non-Salmonella strains. The simulated blood and stool specimen supplemented with S. parayphi A were tested by RT-LAMP assay. Additionally,the blood samples from patients with S. parayphi A and S. typhi infections were detected by RT-LAMP assay. Results The RT-LAMP assay successfully detected hsdM gene of S. parayphi A within 30 to 60 minutes at 65 ℃. Totally 130 S. paratyphi A isolates were detected to be positive while the results of amplification of non-S. paratyphi A isolates were negative. In the detection of purified total RNA from cultured isolates,the detection limit of RT-LAMP was 50 pg per reaction（19 000 copies per reaction）. The sensitivity reached 80 cfu /ml in the extraction of nucleotide from the simulated blood,which was slightly higher than that of real time PCR. For the prepared nucleotide from the simulated stool,the detection limit of RT-LAMP was 500 cfu /g and 0. 8 cfu /g before and after the enrichment of the bacteria,respectively,which was 2- 10 fold higher than that of real time PCR. Regarding the blood samples from patients,the target gene amplification of RT-LAMP were positive in S. paratyphi A infection patients and the detections were negative for S. typhi. Conclusion A RT-LAMP assay was established for the detection of S. paratyphi A,the sensitivity and specificity of the assay was high. The assay established is suitable for the rapid diagnosis of S. paratyphi A infection and identification of the pathogens causing fever with unknown origins.
出处
《疾病监测》
CAS
2014年第9期752-757,共6页
Disease Surveillance
基金
国家科技重大专项(No.2013ZX10004216)~~
