Premium
High‐throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I‐based real‐time PCR combined with melting curve analysis
Author(s) -
TorresCorral Yolanda,
FernándezÁlvarez Clara,
Santos Ysabel
Publication year - 2019
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.13053
Subject(s) - amplicon , biology , primer (cosmetics) , melting curve analysis , sybr green i , real time polymerase chain reaction , polymerase chain reaction , microbiology and biotechnology , 16s ribosomal rna , fish <actinopterygii> , bacteria , gene , genetics , fishery , chemistry , organic chemistry
This work describes a primer pair and a high‐throughput SYBR Green I‐based real‐time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034 × 10 0 amplicon copies per assay (equivalent to 2 × 10 −11 ng/µl, C q value of 30.49 ± 1.71). The developed qPCR protocol allowed the detection of V. salmoninarum in non‐lethal and lethal fish samples with detection levels of 0.17 × 10 0 gene copies in tissues artificially infected and 0.02 × 10 0 in tissues of fish experimentally infected with V. salmoninarum . The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V. salmoninarum in asymptomatic or carrier fish.