Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies

Diseases of Aquatic Organisms

K. H. Wang¹  M. C. Tung¹  C. H. Hu¹  Y. H. Yang¹  Y. L. Hsu¹  T. Hsu¹  C. H. Wang¹  C. F. Lo¹ 

关键词: MBV;    PCR;    ELISA;   

DOI  :  10.3354/dao040093

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

ABSTRACT: The black tiger prawn Penaeus monodon is a valuable aquaculture product in Taiwan. Two specific diagnostic methods were established for P. monodon-type baculovirus, one using polymerase chain reaction (PCR) technology and the otherenzyme-linked immunosorbent assay (ELISA) technology. Monodon-type baculovirus (MBV) was purified by sucrose gradient centrifugation from occlusion bodies of MBV-infected postlarvae of P. monodon. MBV DNA was subsequently purified from theocclusion bodies and its presence was confirmed by PCR using primers of the polyhedrin gene. Based on conserved sequences of the DNA polymerase genes of Autographa californica nuclear polyhedrosis virus (AcMNPV) and Lymantria dispar nuclearpolyhedrosis virus (LdMNPV), primers were designed and synthesized to yield a 714 bp PCR fragment from MBV. However, the sequence of this fragment revealed low homology with that of LdMNPV and AcMNPV. From the DNA sequence of this fragment, a second setof primers was designed, and using these primers, a 511 bp DNA fragment was amplified only when MBV DNA was the template. DNA templates from AcMNPV, white spot syndrome diseased shrimp, or PMO cells (a cell line derived from the Oka organ of Penaeusmonodon) did not give any amplified DNA fragment. Therefore, this primer pair was specific for the diagnosis of MBV. By using intraspleenic immunization of rabbits with purified MBV occlusion bodies, a polyclonal rabbit antiserum against MBV wasobtained. This antiserum could detect nanogram levels of MBV, but did not cross react with white spot syndrome virus (WSSV), homogenates of PMO cells, postlarvae, hepatopancreatic tissue or intestinal tissue of black tiger prawns by competitive ELISA.This sensitive method could detect MBV even in tissue homogenates.

【 授权许可】

Unknown