Design and production of White spot disease (WSSV) multiplex PCR kit

White spot virus disease is one of the diseases that have caused considerable costs to the shrimp industry during the last ۱۵ years. Quickly diagnosing the disease in shrimp hatcher centers and farms by PCR molecular diagnosis kits is one of the ways to prevent the spread of this disease. In this study, different primers were designed based on the sequences of different strains of white spot virus (China, Thailand, Taiwan and Korea) and pathogenic proteins of the virus (VP۲۸, VP۱۹). After optimizing the temperature and adjusting the polymerase chain reaction cycles of each of the designed primers, the produced multiplex kit detected one copy of the white spot virus in the sample with high accuracy, repeatability, and specificity. This product is able to detect the disease within a maximum of ۳.۵ hours from the time of virus DNA extraction from crustacean tissue. The designed multiplex kit contains an M (master mix) and P (primers) buffer along with positive and negative control samples. To identify the virus, briefly, after mixing ۱۵ microliters of buffer M and ۱۴ microliters of buffer P for each sample in sterile ۰.۲ ml micro tubes and adding ۱ microliter of DNA extracted from unknown samples with a concentration of ۲۰۰-۴۰۰ ng/microliter, virus identification, following the amplification of ۶۱۵, ۳۰۰ and ۲۰۰ base per (bp) and or any of them and the sample negative by observing the ۹۹۹ bp band.

Keywords:

White spot virus , multiplex kit , molecular detection , primer , strain