Development and validation of a species-specific loop-mediated isothermal amplification assay for rapid detection of Perkinsus marinus
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Perkinsus marinusis a major protozoan pathogen of oysters, responsible for severe mortality events and substantial economic losses in the global aquaculture industry. Rapid, sensitive, and reliable detection of this parasite is therefore essential for effective monitoring and timely control of dermo disease outbreaks. In this study, we developed and optimized a novel loop-mediated isothermal amplification (LAMP) assay, designated Pm-LAMP, for the specific detection of P. marinus in oyster tissues. The optimized Pm-LAMP assay, employing 5 primers and performed at 67°C, demonstrated high analytical sensitivity, consistently detecting DNA concentrations as low as 40 fg/µl and enabling accurate quantification down to 0.4 pg/µl. The assay exhibited linear amplification across a wide template range from 4 ng/µl to 0.4 pg/µl, with a strong inverse correlation between template concentration and threshold time. Specificity testing confirmed exclusive amplification of P. marinus, with no cross-reactivity observed for P. olseni, P. honshuensis, or P. chesapeaki. This study represents the first LAMP assay specifically designed for the detection of P. marinus. The Pm-LAMP assay was validated using Pacific oyster tissues and cultured P. marinusisolates originating from the USA and Korea and was benchmarked against quantitative real-time PCR (qPCR). Although qPCR exhibited higher sensitivity for detecting trace DNA levels, the Pm-LAMP assay produced results within 20 min while maintaining reliable detection at low DNA concentrations. Diagnostic performance evaluation showed 100% sensitivity and 90.91% specificity, with substantial agreement with qPCR (Cohen’s κ=0.811). Overall, the Pm-LAMP assay provides a rapid, robust, and field-deployable diagnostic tool for P. marinus, supporting improved disease surveillance and sustainable oyster aquaculture management.
Abstract
Perkinsus marinusis a major protozoan pathogen of oysters, responsible for severe mortality events and substantial economic losses in the global aquaculture industry. Rapid, sensitive, and reliable detection of this parasite is therefore essential for effective monitoring and timely control of dermo disease outbreaks. In this study, we developed and optimized a novel loop-mediated isothermal amplification (LAMP) assay, designated Pm-LAMP, for the specific detection of P. marinus in oyster tissues. The optimized Pm-LAMP assay, employing 5 primers and performed at 67°C, demonstrated high analytical sensitivity, consistently detecting DNA concentrations as low as 40 fg/µl and enabling accurate quantification down to 0.4 pg/µl. The assay exhibited linear amplification across a wide template range from 4 ng/µl to 0.4 pg/µl, with a strong inverse correlation between template concentration and threshold time. Specificity testing confirmed exclusive amplification of P. marinus, with no cross-reactivity observed for P. olseni, P. honshuensis, or P. chesapeaki. This study represents the first LAMP assay specifically designed for the detection of P. marinus. The Pm-LAMP assay was validated using Pacific oyster tissues and cultured P. marinusisolates originating from the USA and Korea and was benchmarked against quantitative real-time PCR (qPCR). Although qPCR exhibited higher sensitivity for detecting trace DNA levels, the Pm-LAMP assay produced results within 20 min while maintaining reliable detection at low DNA concentrations. Diagnostic performance evaluation showed 100% sensitivity and 90.91% specificity, with substantial agreement with qPCR (Cohen’s κ=0.811). Overall, the Pm-LAMP assay provides a rapid, robust, and field-deployable diagnostic tool for P. marinus, supporting improved disease surveillance and sustainable oyster aquaculture management.