High‐throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I‐based real‐time PCR combined with melting curve analysis

Abstract

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 × 100 amplicon copies per assay (equivalent to 2 × 10−11 ng/µl, Cq 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 × 100 gene copies in tissues artificially infected and 0.02 × 100 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.

Vagococcus salmoninarum II—qPCR, tropism and egg‐associated transmission

“…Based on initial validation using isolates from the Iron River National Fish Hatchery and tribal hatcheries, the V. salmoninarum qPCR also appears highly sensitive and specific. Additionally, the use of a probe‐based assay, rather than a SYBR Green‐based assay (Torres‐Corral, Fernández‐Álvarez, & Santos, ) decreases chances of non‐specific amplification and allows multiplexing. Though additional validation is necessary, this qPCR appears invaluable for the rapid detection and quantification of V. salmoninarum infections.…”

Development of duplex qPCR targeting Carnobacterium maltaromaticum and Vagococcus salmoninarum

“…Indeed, the ability to co‐detect both these pathogens in a single assay is one advantage conferred using a probe‐based assay rather than an SYBR‐based assay (Torres‐Corral et al., 2019). Importantly, this duplex qPCR assay can be used to quickly differentiate V .…”

Comparative genomics of Streptococcus parauberis: new target for molecular identification of serotype III

“…No amplifications were observed using bacterial suspensions or DNA extracted from other serotypes of S. parauberis or unrelated bacteria. The quantification of the bacterial load was assessed through a standard curve generated by plotting the values of Cq versus the logarithm of the amount (ng) of amplicon as previously described (Fernández-Álvarez et al 2016;Torres-Corral et al 2019). A detection limit of 2.67 × 10 2 copies of the amplicon per μl (equivalent to 3.8 × 10 −9 ng/ μl of genomic DNA) was obtained using the designed qPCR procedure.…”

“…High levels of detection (2.49 × 10 3 -7.37 × 10 2 gene copies) were also observed using this qPCR protocol and DNA obtained from non-lethal fish samples (blood samples) as template, suggesting that this non-lethal diagnostic and typing method could be used without the need for bacterial isolation or the use of anti-S. parauberis sera. Detection levels using DNA extracted from infected fish tissues were lower than those obtained from pure bacterial cultures (bacterial suspensions or DNA extracted from bacterial cultures), possibly due to the presence of host DNA or tissue inhibitors such as hemoglobin or serum proteins (Wiklund et al 2000;Cepeda et al 2003;Fernández-Álvarez et al 2019;Torres-Corral et al 2019). Further studies using a high number of strains isolated from different fish species and other animals in different geographic area will help to determine the serological diversity of S. parauberis and to develop new diagnostic tools.…”