Development of a multiplex real-time enzymatic recombinase amplification assay for differentiation of yellow head virus genotype 1 and 2 in Penaeus vannamei

At present, eight genotypes of yellow head virus (YHV) have been identified, and the main pathogenic genotypes are yellow head virus 1 (YHV1) and gill-associated virus (GAV), which have caused mass death and serious economic losses of shrimp industry. The prevention of YHV complex infection is highly dependent on early and rapid diagnostic, however, the available molecular technologies require complex procedures or expensive equipment. In this study, a multiplex rapid detection method of YHV1 and GAV based on real-time enzymatic recombinase amplification (YHV1/GAV-ERA) was established. Primers and probes were designed to detect the conserved ORF1b gene of YHV1 and GAV. Best primers and probes were selected to effectively amplify the target gene, and the sensitivity was reached 10(2) copies/mu L within 6.17 +/- 0.49 min. While multi-nested PCR could reach the detection limit of 1.9 copies/mu L and 2.1 copies/mu L for YHV1 and GAV by consuming higher time and instrument cost. The YHV1/GAV-ERA can effectively amplify the target gene at 32-45 degrees C within 30 min, and the optimum reaction temperature is 42 degrees C. No cross reaction with white spot syndrome virus (WSSV), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND), Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV) and healthy shrimp DNA were observed. Additionally, the test results of YHV1/GAV-ERA were 100% consistent with the industrial standard multi-nested PCR in shrimp samples. This method can distinguish the two genotypes of YHV and get rid of the dependence on the thermal cycle instrument, and provides a feasible rapid detection method for on-site screening of pathogens.