Rapid detection of Salmonella paratyphi A by reverse transcription loop-mediated isothermal amplification

Objective To establish a rapid assay by reverse transcription loop-mediated isothermal amplification (RT-LAMP) 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 50pg 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.