A new multiplex real-time PCR assay to improve the diagnosis of shellfish regulated parasites of the genus Marteilia and Bonamia

Abstract

Aquaculture including shellfish production is an important food resource worldwide which is particularly vulnerable to infectious diseases. Marteilia refringens, Bonamia ostreae and Bonamia exitiosa are regulated protozoan parasites infecting flat oysters Ostrea edulis that are endemic in Europe. Although some PCR assays have been already developed for their detection, a formal validation to assess the performances of those tools is often lacking. In order to facilitate the diagnosis of flat oyster regulated diseases, we have developed and evaluated a new multiplex Taqman® PCR allowing the detection of both M. refringens and Bonamia sp. parasites in one step. Highlights► M. refringens, B. ostreae and B. exitiosa are regulated parasites of flat oysters. ► First validated PCR detecting all regulated pathogens of flat oysters in one step. ► Multiplex PCR performances similar or higher than currently recommended PCR assays. ► Represents a substantial improvement in time, resources and accuracy. ► Useful to monitors parasites prevalence or for demonstration of freedom.

First characterization of the parasite Haplosporidium costale in France and development of a real‐time PCR assay for its rapid detection in the Pacific oyster, Crassostrea gigas

“…Several conventional (Ko et al 1995, Stokes andBurreson 2001) and multiplex (Penna et al 2001;Russell et al 2004) PCR detecting this parasite exist but the development of a real-time PCR provides several benefits such as a reduced time of analysis, an increased sensitivity and a decreased cross-contamination risk. This real-time PCR presented a detection limit between 2.5 and 5 copies/µL which is similar to other real-time assays developed for other pathogens of molluscs (Polinski et al 2015, Canier et al 2020.…”

“…Meanwhile, the sensitivity varied according to the model and the PCR and was particularly low when no prior was used which was not in agreement with data available in the literature for PCR assays (Aranguren & Figueras, 2016;Caraguel et al, 2012;Ramilo et al, 2013). The addition of prior in the model gave sensitivity values comparable to other PCR or realtime PCR targeting mollusc pathogens (Canier et al, 2020;Gagné et al, 2015;Polinski et al, 2021). The use of prior is generally recommended in Bayesian methods if prior determination is based on independent data and/or based on expert scientific knowledge (Johnson et al, 2018;Cheung et al, 2021).…”

“…2012, Ramilo et al 2013, Aranguren and Figueras 2016. The addition of prior in the model gave sensitivity values comparable to other PCR or real-time PCR targeting mollusc pathogens (Gagné et al 2015, Canier et al 2020, Polinski et al 2021. The use of prior is generally recommended in Bayesian methods if prior determination is based on independent data and/or based on expert scientific knowledge (Johnson et al 2019, Cheung et al 2021…”

“…Two sequences, 1445 and 1354 bp in size, were obtained on the 18S fragment for samples[19][20][21][22][23][24][25][26][27][28][29][30] respectively (GenBank references: MZ666334 and MZ666335). These sequences displayed between 98.31 and 99.45% identities with Haplosporidium costale (AF387122.1) and Haplosporidium sp.…”

Investigating the Environmental Survival of Marteilia refringens, a Marine Protozoan Parasite of the Flat Oyster Ostrea edulis, Through an Environmental DNA and Microscopy-Based Approach

“…For the detection of M. refringens 18S rDNA from dilution range or experimental samples, amplification reactions were carried out using the M. refringens simplex version of the multiplex real-time PCR described in Canier et al (2020).…”