Multiplex Real-time Polymerase Chain Reaction Assays for Simultaneous Detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus

Abstract

ObjectivesA multiplex real-time polymerase chain reaction (RT-PCR) method was developed for the identification of three Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus.MethodsSpecific primers and probes targeting the hlyA, tlh, and vvhA genes were selected and used for multiplex real-time PCR to confirm the identification of V. cholerae, V. parahaemolyticus, and V. vulnificus, respectively. This method was applied to screen Vibrio species from environmental samples and combining it with a culture-based method, its effectiveness was evaluated in comparison with culture-based methods alone.ResultsSpecific PCR fragments were obtained from isolates belonging to the target species, indicating a high specificity of this multiplex real-time PCR. No cross-reactivity with the assay was observed between the tested bacteria. The sensitivity of the multiplex real-time PCR was found to have a lower limit of 104 colony-forming units/reaction for all three Vibrio species. The combination strategy raised the isolation ratio of all three Vibrio species 1.26- to 2.75-fold.ConclusionThis assay provides a rapid, sensitive, and specific technique to detect these three Vibrio species in the environment.

Vibrio cholerae O1 with Reduced Susceptibility to Ciprofloxacin and Azithromycin Isolated from a Rural Coastal Area of Bangladesh

“…Informed consent was obtained from the patients, and parents or legal guardians of the children who participated in this study. Genomic DNA was prepared from the presumptively identified V. cholerae isolates using the boiling lysis method of Park et al ( 2013 ) and V. cholerae species-specific ompW PCR was done to confirm identity of the isolates (Nandi et al, 2000 ).…”

Toxigenic Vibrio cholerae O1 in vegetables and fish raised in wastewater irrigated fields and stabilization ponds during a non-cholera outbreak period in Morogoro, Tanzania: an environmental health study

“…DNA was extracted from colonies picked from TSA by the boiling lysis method. Following removal of cell debris by centrifugation at 12,000 rpm for 3 min, the supernatant containing the template DNA was stored at −20 °C for PCR [ 12 ]. Isolates were confirmed as V. cholerae by PCR for the outer membrane protein encoding gene ( Omp W) [ 13 , 14 ].…”

A label-free multi-functionalized graphene oxide based electrochemiluminscence immunosensor for ultrasensitive and rapid detection of Vibrio parahaemolyticus in seawater and seafood

“…This bacterium has emerged as an important cause of seafood-associated disease outbreaks throughout the world, and is a significant concern for seafood safety [6,7]. Methods available to investigate the presence and quantify VP mainly include most probable number (MPN) [8,9], enzyme-linked immunosorbent assay (ELISA) [10,11], immunofluorescence [12], loop-mediated isothermal amplification (LAMP) [13][14][15][16][17][18], other polymerase chain reaction (PCR) based assays [19][20][21][22][23], DNA probe [24] and sensor-based electrochemical techniques [25]. However, these methods mostly suffer from one or more of the drawbacks such as complex sample pretreatment steps, poor sensitivity, long analytical time, high analytical costs and expensive instrumentation.…”