Development of duplex real time PCR assay for detection of Haplosporidium sp. and Marteilia refringens in shellfish

Abstract:

Two sets of specific oligonucleotide primers for Haplosporidium sp. and Marteilia refringens, along with two TaqMan probes specific for each protozoan parasite were designed with Primer Express 20 software. The reaction parameters such as the concentration of two pair of primers, two TaqMan probes and the reaction buffer were optimized to develop duplex real time PCR assay for the rapid detection of Haplosporidium sp. and Marteilia refringens. The sensitivity of duplex real time PCR assay was 40 template copies for Haplosporidium sp. and Marteilia refringens. The duplex real time PCR assay was found to be specific and to be able to detect and differentiate Haplosporidium sp. and Marteilia refringens, and no positive results were observed when nucleic acid from Perkinsus sp.,Pseudomonas fluorescens, Vibrio parahaemolyticu, V. Alginolyticu, V.Fluvialis and V. Mimicus were used as duplex real time PCR templates. This duplex real time PCR assay is a rapid, sensitive, and specific test for detection of Haplosporidium sp and Marteilia refringens and will be useful for the control of these protozoan parasites in shellfish.