Development of a duplex real-time PCR assay for detection of Perkinsus and Marteilia refringens in shellfish.

Abstract

[Objective] To improve the accuracy and efficiency of the detection which used in Perkinsus sp and Marteilia refringens, and then shorten the detective cycle.[Method]According to the gene sequence of Perkinsus sp and Marteilia refringens from gene bank, design two pairs of specific primers and two TaqMan probes with different fluorophores labeled. Optimizing the reactive conditions and reagent concentration in order that establishing the duplex real-time PCR method for detecting Perkinsus sp and Marteilia refringens simultaneously.[Result] The sensitivity of the duplex real-time PCR method which about Perkinsus sp and Marteilia refringens is 40 template copies. After combine the templates of Perkinsus sp and Marteilia refringens with different concentrations, this method still could be detect this two protozoan efficiently and synchronously.[Conclusion] The established duplex real-time PCR method for detecting Perkinsus sp. and Marteilia refringens possesses lots of advantages, such as specific, sensitive, rapid, quantitative and reproducible, can be used for clinical detection of infection which was caused by Perkinsus sp. and Marteilia refringens.