Exploration and application of detection and genotyping standards for white spot syndrome virus

White spot syndrome virus (WSSV) is the pathogen that causes white spot disease (WSD) in shrimp. The mortality rate of infected shrimp can reach 100% in one week. Up to now, both the Office International Des Epizooties (OIE) and the national standard have adopted the same universal primers for nested PCR. With increasing number of WSSV sequences reported, the recommended nested PCR has been found at risk of missing detection. Therefore, a new nested PCR to detect WSSV in crustaceans in our laboratory has been developed. Two sets of detection primers for nested PCR were designed. The sensitivity of the first-step PCR of the two sets of redesigned detection primers were 10 times greater than that of the universal primers. We selected JC-F/JC-R primers for the follow-up experiments. In the detection of WSSV in crustaceans caught in beach in Yantai, there were no results in the first-step PCR with both the universal primers and JC-F1/JC-R1. In the second-step PCR, the detection rate of universal primers was 23.75%, and that of our redesigned detection primers (JC-F2/JC-R2) was 65%. In order to promote the study of molecular epidemiology of WSSV in the future, we also designed universal primers for ORF14/15 and ORF75. The samples of Procambarus clarkii carried WSSV collected from Hubei and Zhejiang were analyzed. According to the sequence information of ORF14/15, they all belong to type I. The genotypes of ORF75 of them were all the M-f-c. We speculated that genotype M-f-c might evolve from subtype II, and there may be multiple ways of evolution of the M-f-c.