Screening the post-larvae of Macrobrachium rosenbergii for early detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) by RT-PCR and immunological techniques.

摘要

White tail disease (WTD) was found to be a serious problem in hatcheries and nursery ponds of freshwater prawn (Macrobrachium rosenbergii). The causative organisms have been identified as Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). RT-PCR and immunological techniques such as Western blot and ELISA were used for early detection of MrNV and XSV in post-larval samples obtained from time-course experiments at different time intervals. Two viruses were purified from diseased post-larvae of M. rosenbergii by a combination of low and high speed centrifugation using sucrose and CsCl gradients to raise the antisera separately. One structural protein with molecular weight of 43 kDa (CP-43) was identified from the purified preparation of MrNV, and two overlapping polypeptides of about 17 kDa (CP-17) and 16 kDa (CP-16) were found in XSV particles by SDS-PAGE. The antisera raised against CP-43 of MrNV, CP-16 and CP17 of XSV in mice were used to detect MrNV and XSV by Western blot and ELISA. Published primers specific to MrNV and XSV were used for the early detection of these viruses by RT-PCR and nested RT-PCR. The post-larval samples collected at 3 h post infection (h p.i.) showed positive for both viruses by nested RT-PCR and negative by RT-PCR, Western blot and ELISA techniques. The samples collected at 24 h p.i. and thereafter were found to be positive for MrNV and XSV by RT-PCR, ELISA and Western blot analyses.