Establishment of RT-LAMP assay to detect Salmonella Typhi in blood

Objective To establish reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect Salmonella Typhi in blood. Methods A set of six specific primers recognizing distinct regions of fliC-d gene of S. Typhi 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 in 48 serotypes, the non-Salmonella strains causing diarrhea and the strains of 8 bacteria inducing bacteremia with fever as main symptom. The simulated blood specimens supplemented with S. Typhi were tested with RT-LAMP assay, and the detection limit of RT-LAMP was compared with that of real-time PCR. Results The RT-LAMP assay successfully detected fliC-d gene of S. Typhi within 30 to 60 minutes at 65 ℃. Totally 44 strains of S. Typhi were detected to be positive. Except 4 rare serotypes of non-salmonella were positive in amplification, the amplification of the strains of non-Salmonella in 30 serotypes, the strains of 5 pathogens causing diarrhea and the strains of 8 bacteria causing bacteremia with fever as main symptom were negative. In the detection of total RNA from pure cultured isolates, the detection limit of RT-LAMP was 0.5 pg per reaction (97copies per reaction). In the extracting nucleotide from simulated blood, the sensitivity was 1 cfu /ml, which was 100 fold higher than that of real time PCR. Conclusion The RT-LAMP assay for detecting S. Typhi with high sensitivity and specificity was established, which would be suitable for rapid diagnosis of S. Typhi infection and identifying pathogen of fever with unknown origin , and can be used in early diagnosis, prevention/control and treatment of typhoid fever.