Design and Validation of a Novel Multiplex Real-Time PCR Assay for Vibrio Pathogen Detection

Abstract

Three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus--account for the majority of vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species. Additionally, biochemical identification and confirmation require 2 or more days to complete. Rapid and sensitive molecular techniques for the detection of vibrio pathogens would be useful for the surveillance and management of outbreaks. To facilitate the identification of human-pathogenic species, we designed and validated a highly sensitive, specific, and robust multiplex real-time PCR assay to identify V. cholerae, V. parahaemolyticus, and V. vulnificus using a four-dye configuration in a convenient lyophilized format. Multiple Vibrio strains were sequenced to verify candidate target TaqMan sites. Several individual assays within the multiplex contain multiple primers or probes to ensure detection of polymorphic variants. V. cholerae, V. parahaemolyticus, and V. vulnificus were detected either individually or in mixtures at ≤30 genomic copies. V. cholerae was specifically detected in the presence or absence of Vibrio mimicus. The Vibrio multiplex assay showed 100% specificity to all targets analyzed and no detection of nearest neighbor strains. Each assay exhibited 100% ± 10% efficiency. Multiplex real-time PCR can simplify pathogen detection and reduce costs per test since three species can be analyzed in a single reaction tube. Rapid and accurate detection of pathogenic vibrios in shellfish or seawater samples will improve the microbiological safety of seafood for consumers.

Vibrio cholerae and Cholera biotypes

“…These methods have high sensitivity, specificity, and speed. The methods used for V. cholerae detection by conventional PCR and/or real-time PCR are well established (Wang et al, 2007;Tebbs et al, 2011;Teklehaimanot et al, 2014). Many research studies have identified Vibrio cholerae strains using 16S rRNA sequencing, pulsed-field gel electrophoresis (PFGE), nested and multiplex PCR (Chakraborty et al, 1999;Kong et al, 2002;Tarr et al, 2007;Mendes et al, 2008;Keshav et al, 2010).…”

Multipurpose assessment for the quantification of Vibrio spp. and total bacteria in fish and seawater using multiplex real‐time polymerase chain reaction

“…The goal of the current study was to develop and evaluate a multiplex real‐time PCR assay that would provide a rapid, sensitive, and quantitative method for the detection of V. anguillarum , V. parahaemolyticus , V. vulnificus , and total bacteria in fish fillets and seawater. While there exist methods to detect V. cholerae, V. parahaemolyticus , and V. vulnificus using multiplex real‐time PCR, there are no methods to detect V. anguillarum and the other Vibrio spp. at the same time.…”

Simultaneous quantification of the most common and proteolytic Pseudomonas species in raw milk by multiplex qPCR

“…For example, multiplex qPCR assays have been developed for the detection of Salmonella spp., Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Campylobacter spp., or Vibrio spp. in various foods (Hong et al 2007;Tebbs et al 2011;Forghani et al 2016;Liu et al 2017;Heymans et al 2018;Parichehr et al 2019). In addition, several applications deal with probiotic or beneficial organisms, such as yeasts, Acetobacter spp., or different lactic acid bacteria (LAB) in kefir or starter cultures in cheese production (Bottari et al 2013;Nejati et al 2020).…”