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Development of a multiplex PCR assay for rapid and simultaneous detection of four genera of fish pathogenic bacteria
Author(s) -
Zhang D.F.,
Zhang Q.Q.,
Li A.H.
Publication year - 2014
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.12303
Subject(s) - biology , microbiology and biotechnology , aeromonas hydrophila , aeromonas , pathogenic bacteria , bacteria , edwardsiella tarda , vibrio , streptococcus iniae , multiplex polymerase chain reaction , enterococcus , amplicon , polymerase chain reaction , antibiotics , gene , genetics
Species of genus A eromonas , V ibrio , E dwardsiella and S treptococcus are the most common fish pathogenic bacteria that cause economically devastating losses in aquaculture. A multiplex polymerase chain reaction (m PCR ) was developed for the simultaneous detection and differentiation of the four genera of fish pathogenic bacteria. Through the use of genus‐specific primers instead of species‐specific ones, the current m PCR covered much more target bacterial species compared with previously reported species‐specific m PCR methods. The specificity of the four putative genus‐specific primers was validated experimentally while used exclusively (uniplex PCR ) or combined (m PCR ) against bacterial genomic DNA templates of the target bacteria and nontarget bacteria. The PCR amplicons for the following genera were obtained as expected: A eromonas (875 bp), Vibrio (524 bp), Edwardsiella (302 bp) and Streptococcus (197 bp), and the fragments could be separated clearly on the agarose gel electrophoresis. The m PCR did not produce nonspecific amplification products when used to amplify 21 nontarget species of bacteria. The m PCR detection limits for each target bacterial genera were 50 colony‐forming units ( CFU ) in pure culture and 100 CFU in fish tissue samples. In conclusion, the m PCR assay was proven to be a powerful alternative to the conventional culture‐based method, given its rapid, specific, sensitive and reliable detection of target pathogens. Significance and Impact of the Study The fish pathogenic bacteria of genus A eromonas , V ibrio , E dwardsiella and S treptococcus frequently cause severe outbreaks of diseases in cultured fish, and the genus‐specific multiplex PCR assay developed in this study can detect the bacteria of the four genera when present in the samples either alone or mixed. The m PCR assay is expected to identify the causative agents more efficiently than uniplex PCR or species‐specific multiplex PCR for clinical diagnosis, resulting in the earlier implementation of control measures. This m PCR assay provides a rapid, specific and sensitive tool for the detection or identification of common fish pathogenic bacteria in aquaculture practice.