Non-Protein Coding RNA Genes as the Novel Diagnostic Markers for the Discrimination of Salmonella Species Using PCR

Abstract

Salmonellosis, a communicable disease caused by members of the Salmonella species, transmitted to humans through contaminated food or water. It is of paramount importance, to generate accurate detection methods for discriminating the various Salmonella species that cause severe infection in humans, including S. Typhi and S. Paratyphi A. Here, we formulated a strategy of detection and differentiation of salmonellosis by a multiplex polymerase chain reaction assay using S. Typhi non-protein coding RNA (sRNA) genes. With the designed sequences that specifically detect sRNA genes from S. Typhi and S. Paratyphi A, a detection limit of up to 10 pg was achieved. Moreover, in a stool-seeding experiment with S. Typhi and S. Paratyphi A, we have attained a respective detection limit of 15 and 1.5 CFU/mL. The designed strategy using sRNA genes shown here is comparatively sensitive and specific, suitable for clinical diagnosis and disease surveillance, and sRNAs represent an excellent molecular target for infectious disease.

Insights from Comparative Genomics of the Genus Salmonella

“…That most of the Salmonella genomes are nicely clustered according to their serotype in Figure 7 is surprising, as the nonprotein coding sRNA genes analyzed here do not have a speciic role in expression of surface antigens. The correlation identiied here is in line with a publication that sRNA genes can be used as targets for serotype-speciic PCR detection of Typhi and Paratyphi [23]. It was recently described that some sRNA genes of S. Typhimurium are under regulation of Sigma 28, and there is extensive cross talk between genes of the Salmonella pathogenicity pathways SPI1 and SPI2 and particular sRNA genes [24].…”

One-Step PCR Detection of Salmonella Pullorum/Gallinarum Using a Novel Target: The Flagellar Biosynthesis Gene flhB

“…The target fragment was amplified at concentrations of 58.5 ng/μL to 5.85 pg/μL DNA ( Figure 3A ). The results suggested that at least 5.85 pg/μL of genomic DNA was required for detection of S. Pullorum using this assay, which was slightly lower than previous studies (10 pg/μL) using Salmonella genomic DNA (Nithya et al, 2015). On the other hand, a 10-fold serial dilution of S. Pullorum cells that ranged from 10 4 CFU to 10 -1 CFU per PCR system was evaluated.…”

Characterization of Different Molecular Markers for Identification of Salmonella Enterica Serovar Typhi in Pakistani Population

“…In current study, StyR-36 gene was amplified in 40 samples showing 68.97% amplification results. Nithya et al (2015) conducted a study on Salmonella species by targeting StyR-3, StyR-36 and StyR-143 genes in order to evaluate the specificity of genes in S. paratyphi and S. typhi. StyR-3 and StyR-46 was found in both the species while StyR-36 was amplified only in S. typhi.…”

“…Extracted gDNA was confirmed on 1% agarose gel and qualitatively and quantitatively analyzed using spectrophotometer (HP 8451A Diode Array Spectrophotometer, USA). Two pair of primers, i-e, ST1/ST2 (5'-TATGCCGCT ACATATGATGAG-3', 5'-TTAACGCAGT AAAGAGAG-3') and StyR-36F/StyR-36R (5'-TGCCATGTAATCGGACGCCGAC-3', 5'-AGCCAACAAACGCGGTTGCG-3') were used to amplify 498 bp of flagellin and 204 bp of StyR-36 gene (Haque et al, 1999;Nithya et al, 2015), respectively. Amplification of respective genes was carried out using PCR Sprint Thermal Cycler (Thermo Electron Corporation, USA).…”

“…In developing countries like Pakistan, the infectious agents are serologically diagnosed which is usually considered as an unreliable method. Molecular techniques are the sole resolving methods to accurately diagnose Salmonella enterica serovar Typhi by targeting the specific genes i-e, flagellin and/or StyR-36 genes (Lamkanfi et al, 2007;Nithya et al, 2015).…”