In this study,a triplex real-time PCR method was developed for the simultaneous detection of decapod iridescent virus 1(DIV1),enterocytozoon hepatopenaei(EHP)and infectious precocious virus(IPV)in shrimp,based on specific primers and TaqMan probes respectively targeting DIV1 MCP gene,EHP SSU gene and IPV polyprotein A gene.The method was able to specifically detect DIV1,EHP and IPV simultaneously without cross-reactions with other main shrimp pathogens such as white spot syndrome virus(WSSV),Vibrio parahaemolyticus(AHPND),infectious hypodermal and haematopoietic necrosis virus(IHHNV),covert mortality nodavirus(CMNV),infectious myonecrosis virus(IMNV),taura syndrome virus(TSV),yellow head virus 1(YHV-1).The method had a detection limit of 1.67 × 101 copies/μL from the sensitivity test results of 10 times diluted plasmids mixture of DIV1,EHP and IPV,slightly lower than that of the reference single real-time PCR for each pathogen with a detection limit of 1.67 × 100 copies/μL.Three concentrations of pDIV1,pEHP and pIPV(5 × 105 copies/μL,5 × 104 copies/μL,5×103 copies/μL)were selected to evaluate the repeatability of the method,and the results showed the coefficient of variation in both intra-and inter-group repeatability tests was less than 1％,indicating that the method had good repeatability.The detection results from 105 samples of fry and brood shrimp by the triplex method showed the positive rate of DIV1,EHP and IPV was 21.9％(23/105),14.3％(15/105)and 45.7％(48/105)respectively,and the mixed infection rate of the three pathogens was 1.9％(2/105),which was consistent with the detection results by DIV1,EHP and IPV single fluorescence quantification method.The coincidence rate of positive samples was 100％.The method established in this study could provide a rapid,sensitive and specific technique for simultaneous detection of DIV 1,EHP and IPV in clinical samples from daily monitoring and epidemiological investigation.
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