Nested polymerase chain reaction and in situ hybridization for detection of nucleopolyhedrosis

A nested polymerase chain reaction (PCR) and in situ hybridization were developed for detection of baculoviruses in insects or other arthropods with nucleopolyhedrosis. The nested PCR was based on the sequences of polyhedrin genes from baculoviruses. Two sets of primers were designed, primers set, 35/36, was for the first step of amplification and yielded a product of around 680 bp, the second primer, 35-1/36-1, was designed to yield a product of around 335bp from the fragment amplified by the first primer set. The sensitivity of this two-step amplification was 100 to 1000 times higher than that of the one-step amplification by primer set (35/36). Samples which contained baculovirus DNA yielded an amplification product showing the expected DNA fragment mobility, whereas nucleic acid extracted from tissue samples of clinically healthy insects or uninfected cells showed no such DNA fragment, thereby confirming the specificity of the primers. Using the 35/36 amplicon as a probe, the PenuNPV-infected cells show positive reaction by in situ hybridization. Two-step DNA amplification and in situ hybridization with the DNA probe developed in the present paper provide effective detection and diagnostic tools for screening insects or other arthropods, especially crustacean species, crabs and shrimps, for baculovirus infections, and may be important in preventing (and/or controlling/enhancing) the infection of baculoviruses.