Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams

Abstract

The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD) in the Manila clam Venerupis (=Ruditapes) philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification of V. tapetis strains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600(T) V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW) or extrapallial fluids (EF) samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 10(1) bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes.

Keywords: Brown ring disease; Marine pathogen; Molecular diagnostic; Taqman real-time PCR; Venerupis philippinarum; Vibrio tapetis; virB4 gene.

Figure 1. Schematic view of the infection procedure.

Figure 2. Photography of (A) BRD- clam and (B) BRD+ clam.

From Richard et al., 2015, unpublished data.

Figure 3. Visualization of the PCR product in agarose gel obtained with qPCR virB4 assay for representative strains of Vibrio, i.e., which were tested positive and negative for BRD development after an infection experiment.

Lanes MT corresponds to the BenchTop DNA ladder (Promega, Madison, WI, USA). T-H₂O represents the water negative control.

Figure 4. Standard curve for the detection and quantification of the virB4 gene by Taqman real-time PCR, in dilution range of EF samples artificially spiked with CECT4600^T bacterial strain.

Standard curve was generated by plotting the log cell number of bacteria present in PCR DNA template against Ct values.

Figure 5. Kinetics of clam infection by CECT4600^T V. tapetis strain by virB4 real-time PCR in extrapallial fluids sampled at 0, 1, 2, 7, 14 and 30 days post-injection.

0d means not injected. * corresponds to BRD+ clam.

Figure 6. Kinetics of non-injected clams by virB4 real-time PCR in extrapallial fluids sampled at 0, 1, 2, 7, 14 and 30 days of sampling during the experiment.

* corresponds to BRD+ clam.

Figure 7. Kinetics of FSW-injected clams by virB4 real-time PCR in extrapallial fluids sampled at 0, 1, 2, 7, 14 and 30 days of sampling during the experiment. 0d means not injected.

* corresponds to BRD+ clam.