Utilization of Small RNA Genes to Distinguish Vibrio cholerae Biotypes via Multiplex Polymerase Chain Reaction

Abstract

The diarrheal disease "cholera" is caused by Vibrio cholerae, and is primarily confined to endemic regions, mostly in Africa and Asia. It is punctuated by outbreaks and creates severe challenges to public health. The disease-causing strains are most-often members of serogroups O1 and O139. PCR-based methods allow rapid diagnosis of these pathogens, including the identification of their biotypes. However, this necessitates the selection of specific target sequences to differentiate even the closely related biotypes of V. cholerae. Oligonucleotides for selective amplification of small RNA (sRNA) genes that are specific to these V. cholerae subtypes were designed. The resulting multiplex PCR assay was validated using V. cholerae cultures (i.e., 19 V. cholerae and 22 non-V. cholerae isolates) and spiked stool samples. The validation using V. cholerae cultures and spiked stool suspensions revealed detection limits of 10-100 pg DNA per reaction and 1.5 cells/mL suspension, respectively. The multiplex PCR assay that targets sRNA genes for amplification enables the sensitive and specific detection, as well as the differentiation of V. cholerae-O1 classical, O1 El Tor, and O139 biotypes. Most importantly, the assay enables fast and cheaper diagnosis compared with classic culture-based methods.

Molecular detection of 7SL‐derived small RNA is a promising alternative for trypanosomosis diagnosis

“…Over the last few decades, non-invasive tools have been developed for detecting cell-free miRNA for cancer diagnosis and monitoring relapses (Kroh, Parkin, Mitchell, & Tewari, 2010;Yu et al, 2008). New strategies using microRNAs as diagnostic targets for infectious diseases are beginning to develop for bacterial or viral infections (Ahmed et al, 2019;Vivek, Zahra, & Kumar, 2019), and trypanosomes have a large repertoire F I G U R E 2 Stability of 7SL-sRNA in equine sera after storage at different temperatures for 7 days. Two sera: (a) medium positive serum and (b) strongly positive serum were incubated at three different temperatures: 4°C (blue), room temperature (green) and 30°C (orange) for 7 days.…”

“…Over the last few decades, non‐invasive tools have been developed for detecting cell‐free miRNA for cancer diagnosis and monitoring relapses (Kroh, Parkin, Mitchell, & Tewari, 2010; Yu et al., 2008). New strategies using microRNAs as diagnostic targets for infectious diseases are beginning to develop for bacterial or viral infections (Ahmed et al., 2019; Vivek, Zahra, & Kumar, 2019), and trypanosomes have a large repertoire of small sRNAs that could be potentially used for trypanosomosis diagnosis (Michaeli et al., 2012). In this context, the report by Chiweshe et al.…”

Advances in cholera research: from molecular biology to public health initiatives

“…Several diagnostic methods have been developed to distinguish between Classical and El Tor biotypes in recent years. PCR-based genotypic assays typically screen for specific sequence variations in virulence genes including tcpA , ctxA , ctxB , and toxR , while another genome-based method targets unique small RNA genes ( Crumfield et al, 2018 ; Greig et al, 2018 ; Ahmed et al, 2019 ). Biotype can be distinguished phenotypically by evaluating antibiotic and phage susceptibility, capability for hemolysis and proteolysis, and variations in metabolism of citrate and glucose ( Crumfield et al, 2018 ; Lee et al, 2020 ).…”