Loop-mediated isothermal amplification for diagnosing marine pathogens in tissues of Crassostrea spp. and white shrimp, Litopenaeus vannamei, farmed in Mexico
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Abstract
Loop-mediated isothermal amplification (LAMP) is an accurate, sensitive, rapid, and easy-to-perform method for gene amplification under isothermal conditions, and it has served as a powerful diagnostic tool. In this study, we used LAMP to develop a diagnostic protocol for detecting Vibrio parahaemolyticus and white spot syndrome virus in whiteleg shrimp, and Perkinsus spp. in Crassostrea spp. in Mexico. These pathogens are associated with different diseases and are considered a threat in the aquaculture industry. Infected and uninfected oysters and shrimp were obtained from farms in the northwest coast of Mexico to standardize the LAMP assay. We determined the candidate target genes in the first-round analysis of many sets of primers, and then we chose a set of primers that successfully amplified with Mexican samples. We optimized the LAMP reactions for each pathogen with the chosen primer sets using temperature gradients from 61 to 65 ºC, DNA concentrations from 2.5 pg to 250.0 ng, and reaction times from 10 to 60 min. This study established a diagnostic procedure for detecting pathogens in oysters and shrimp from Mexico. Early diagnosis and treatment of pathogens can immensely reduce disease transmission in aquaculture farms.
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