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Development of a two‐step, non‐probed multiplex real‐time PCR for surveilling V ibrio anguillarum in seawater
Author(s) -
Hickey M E,
Richards G P,
Lee JL
Publication year - 2015
Publication title -
journal of fish diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.819
H-Index - 85
eISSN - 1365-2761
pISSN - 0140-7775
DOI - 10.1111/jfd.12264
Subject(s) - vibrio anguillarum , biology , microbiology and biotechnology , seawater , primer (cosmetics) , multiplex , multiplex polymerase chain reaction , real time polymerase chain reaction , vibrio , pathogen , polymerase chain reaction , bacteria , chemistry , gene , ecology , biochemistry , genetics , organic chemistry
Abstract Vibrio anguillarum is an aggressive and halophilic bacterial pathogen most commonly originating from seawater. V ibrio anguillarum presence in fisheries and aquaculture facilities causes significant morbidity and mortality among aquaculture species primarily from haemorrhaging of the body and skin of the infected fish that eventually leads to death, collectively recognized as the disease vibriosis. This study served to develop a non‐probe, multiplex real‐time PCR assay to rapidly detect V. anguillarum presence in seawater. Specific primers targeting genes vah 1, emp A and rpo N of V. anguillarum were selected for multiplex reaction among 11 different primer sets and the extension step was eliminated. Primer concentration, denaturation time as well as annealing time and temperature of DNA amplification were optimized, thus reducing reaction duration. The two‐step, non‐probed multiplex real‐time PCR set forth by this study detects as little as 3 CFU mL −1 of V . anguillarum presence in sea water, without enrichment cultivation, in 70 min with molecular precision and includes melting curve confirmation.