Purification and amplification of DNA from Penaeus monodon-type baculovirus (MBV)

The purpose of this paper was to purify and amplify the DNA fragment of Penaeus monodon-type baculovirus (MBV). Using 30-50% caesium chloride gradients, MBV virions and occlusion bodies with density parameters of 1.28-1.29 and 1.32-1.33 g/ml, respectively, were purified. Two oligonucleotide primers have been successfully designed and utilized for the amplification of a DNA fragment of MBV. After 35 amplification cycles of the MBV DNA fragment, a large amount of amplified product with an approximate molecular weight of 600 bp was obtained. This is the first successfully published work on the amplification of MBV using the polymerase chain reaction (PCR). Using the same primers, DNA extracted from MBV noninfected P. monodon, P. japonicus, and P. orientalis had a negative PCR response. However, a positive PCR response was obtained from DNA extracted from MBV-infected postlarval P. monodon. DIG-dot blot hybridization technique using PCR product obtained from the present study as a probe further confirmed that the product is originated from a portion of MBV polyhedrin gene. It is also suggested that PCR product may be beneficial for an accurate and early diagnosis of MBV infection in larval shrimp.