Development of a loop-mediated isothermal amplification method for detection of Perkinsus spp. in mollusks

Abstract

Perkinsus is a genus of unicellular protozoan parasite responsible for mass mortality of several commercially valuable mollusks. Surveillance and inspection of its epidemiology in the field calls for convenient and rapid detection methods. Here, a loop-mediated isothermal amplification (LAMP) assay was developed to detect the presence of Perkinsus spp. in mollusks. Specific LAMP primers were designed targeting the conserved internal transcribed spacer 2 (ITS-2) region of the rRNA gene of Perkinsus spp. Using ITS-2 recombinant plasmid as a template, we optimized the LAMP reaction system and conditions and then evaluated the analytical sensitivity and specificity of the assay. The LAMP assay was validated using clam samples collected from coastal areas in eastern China and oysters imported to China and compared with the traditional Ray's fluid thioglycollate culture method (RFTM). Our results showed that the LAMP detection method for Perkinsus was successful. The detection limit was 10 copies of plasmid DNA. Compared to the RFTM assay, the LAMP detection method was more sensitive (56 versus 52 positive out of 60 samples). P. olseni and P. marinus from infected hosts were successfully detected by this method. The LAMP method is rapid, sensitive, and specific for Perkinsus spp. detection, and could be used to screen for perkinsosis both on farms and at ports.