Primer sets and multiplex detection methods for simultaneous qualitative and quantitative analysis of EHP, DIV1, IHHNV, WSSV, and VAHPND in crustaceans

A detection method of multiplex fluorescent quantitative detection method for simultaneous qualitative and quantitative detection of multiple pathogens, wherein, the detection method is non-diagnostic and non-therapeutic, wherein the detection method is applied in rapid pathogen detection in aquatic animals;

• the pathogens are Enterocytozoon hepatopenaei, (EHP), Decapod iridescent virus 1, (DIV1), infectious hypodermal and hematopoietic necrosis virus, (IHHNV), white spot syndrome virus, (WSSV), and acute hepatopancreatic necrosis disease causing Vibrio, (VAHPND), the detection method comprises steps of:
• (1) collecting samples and extracting genomic DNA as a template for fluorescent quantitative reactions, wherein a reaction system for fluorescent quantitative PCR has a total volume of 25 μL, and the reaction system comprises 2×dsDNA-binding fluorescent dye premix containing 50 U/μL Taq DNA polymerase, 2 mM dNTPs, 50× 5-carboxy-X-rhodamine reference dye, dsDNA-binding fluorescent dye, 20 mM Tris-HCl, 0.1 mM EDTA, 100 mM NaCl, 0.5% polysorbate 20, 1 mM dithiothreitol, 50% volume ratio of glycerol:
• 12.5 μL;
• primers:
• 0.56 μM of EHP-2-F, 0.56 μM of EHP-2-R, 0.40 μM of IHHNV-P-3F, 0.40 μM of IHHNV-P-3R, 0.40 μM of WSSV-Y-2F, 0.40 μM of WSSV-Y-2R, 0.32 μM of DIV1-M-4F, 0.32 μM of DIV1-M-4R, 0.32 μM of VP-PB-3F, and 0.32 μM of VP-PB-3R;
• 1 μL of genomic DNA template;
• and deionized water being added to a total volume to 25 μL;
• (2) mixing primers with dsDNA-binding fluorescent dye premix and the genomic DNA to form an amplification reaction system, then performing fluorescent quantitative amplification, wherein the amplification reaction system was loaded into a real-time PCR instrument, and the fluorescent quantitative reaction conditions are:
• 95° C. for 3 minutes, 40 cycles of 95° C. for 10 seconds and 64° C. for 20 seconds;
• (3) after amplification, analyzing melting curves to determine the presence of EHP, DIV1, IHHNV, WSSV, and VAHPND in the samples, wherein the analyzing melting curves was conducted at 95° C. for 15 seconds, and 64° C. for 1 minute, and melting curve data was collected at 68° C. to 85° C.;
• wherein the method further comprises constructing recombinant plasmids containing gene sequences corresponding to a SWP protein of EHP, a MCP protein of DIV1, a NS1 protein of IHHNV, a WSV313 protein of WSSV, and a PirB protein of VAHPND;
• wherein sequences of the primers are SEQ ID NOs.:
• 1-10.