Biocomputational Identification of sRNAs in Leptospira interrogans Serovar Lai.

Regulatory small RNAs (sRNA) are RNA transcripts that are not translated into proteins but act as functional RNAs. Pathogenic Leptospira cause an epidemic spirochaetal zoonosis, Leptospirosis. It is speculated that Leptospiral sRNAs are involved in orchestrating their pathogenicity. In this study, biocomputational approach was adopted to identify Leptospiral sRNAs. In this study, two sRNA prediction programs, i.e., RNAz and nocoRNAc, were employed to screen the reference genome of Leptospira interrogans serovar Lai. Out of 126 predicted sRNAs, there are 96 cis-antisense sRNAs, 28 trans-encoded sRNAs and 2 sRNAs that partially overlap with protein-coding genes in a sense orientation. To determine whether these candidates are expressed in the pathogen, they were compared with the coverage files generated from our RNA-seq datasets. It was found out that 7 predicted sRNAs are expressed in mid-log phase, stationary phase, serum stress, temperature stress and iron stress while 2 sRNAs are expressed in mid-log phase, stationary phase, serum stress, and temperature stress. Besides, their expressions were also confirmed experimentally via RT-PCR. These experimentally validated candidates were also subjected to mRNA target prediction using TargetRNA2. Taken together, our study demonstrated that biocomputational strategy can serve as an alternative or as a complementary strategy to the laborious and expensive deep sequencing methods not only to uncover putative sRNAs but also to predict their targets in bacteria. In fact, this is the first study that integrates computational approach to predict putative sRNAs in L. interrogans serovar Lai. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01050-9.

Identification of Five Novel Salmonella Typhi-Specific Genes as Markers for Diagnosis of Typhoid Fever Using Single-Gene Target PCR Assays.

Salmonella Typhi (S. Typhi) causes typhoid fever which is a disease characterised by high mortality and morbidity worldwide. In order to curtail the transmission of this highly infectious disease, identification of new markers that can detect the pathogen is needed for development of sensitive and specific diagnostic tests. In this study, genomic comparison of S. Typhi with other enteric pathogens was performed, and 6 S. Typhi genes, that is, STY0201, STY0307, STY0322, STY0326, STY2020, and STY2021, were found to be specific in silico. Six PCR assays each targeting a unique gene were developed to test the specificity of these genes in vitro. The diagnostic sensitivities and specificities of each assay were determined using 39 S. Typhi, 62 non-Typhi Salmonella, and 10 non-Salmonella clinical isolates. The results showed that 5 of these genes, that is, STY0307, STY0322, STY0326, STY2020, and STY2021, demonstrated 100% sensitivity (39/39) and 100% specificity (0/72). The detection limit of the 5 PCR assays was 32 pg for STY0322, 6.4 pg for STY0326, STY2020, and STY2021, and 1.28 pg for STY0307. In conclusion, 5 PCR assays using STY0307, STY0322, STY0326, STY2020, and STY2021 were developed and found to be highly specific at single-gene target resolution for diagnosis of typhoid fever.