Identification of Salmonella Enterica Serovar Typhi DNA by loop-mediated isothermal amplification with fluorescent detection

Abstract

Introduction. Real-time PCR may be used along with loop isothermal amplification method (LAMP) allowing to conduct the study in 30–40 minutes to diagnose typhoid fever. Several LAMP assay variations for detecting Salmonella enterica serovar Typhi have been described. The studies report that relevant primers were tested on strains specific for Malaysia and China. We attempted to evaluate the LAMP primers described above for identifying S. Typhi strains specific for the Russian Federation and compare their sensitivity and specificity with each other. Materials and methods. A comparative in silico analysis of target sequences was carried out both in the open NCBI database and among the genetic sequences of collection strains at the St. Petersburg Pasteur Institute. Several sets of primers for LAMP amplification of various S. Typhi genome regions were tested. The tested primers amplify the following fragments: SalTyp1 — region of the STY1607 gene, SalTyp2 — region of the STY2879 gene, SalTyp3 — region of the STBHUCCB_38510 gene of the PapD chaperone. Results. In silico analysis of LAMP primers showed that only the SalTyp 3 set has strict specificity for Salmonella enterica serovar Typhi. For the SalTyp 1 and SalTyp 2 an opportunity of false-positive reactions with some E. coli strains was shown. A LAMP method for DNA fluorescent detection of the typhoid fever causative agent was chosen. Assay has been based on a marker gene termed STBHUCCB_38510 and amplification with six specific primers. The detection limit was 20 copies/reaction in reference plasmids and the reaction time lasted for 35 minutes. The specificity of the method was tested on DNA specimens of 20 S. Typhi isolates and 90 strains of other heterologous bacteria from 24 different species. No false positive or false negative results were identified. Conclusion. The developed method can be used in clinical practice for laboratory confirmation of typhoid fever diagnosis as a part of epidemiological monitoring of environmental objects as well as food products.