Polymerase chain reaction (PCR) amplification of bacilliform virus (RV-PJ) DNA in Penaeus japonicus Bate and systemic ectodermal and mesodermal baculovirus (SEMBV) DNA in Penaeus monodon Fabricius.

摘要

Genomic DNA fragments of RV-PJ (rod-shaped nuclear virus of P. japonicus) digested with EcoRI-HindIII were inserted into the EcoRI-HindIII site of the plasmid pGEM3zf(+) and the recombinant plasmids were used to transform Escherichia coli XL1-BlueMRF. Among the transformed cells, a clone with a plasmid having an insert of 889 bp was used to construct a pair of primers. The primers, each consisting of 20 nucleotides, were designed to amplify a 643 bp sequence. A single band of a 643 bp DNA fragmentwas observed after 30 cycles of PCR amplification of the viral genomic DNA of RV-PJ and SEMBV. Tissues removed from infected and uninfected shrimps were used for the DNA extraction for the PCR templates. DNA of lymphoid organs infected with RV-PJ and SEMBV formed a single band that appeared at the same position as the genomic DNA. No PCR product was obtained from DNA from organs infected with baculoviral mid-gut gland necrosis virus, yellow-head disease virus or Baculovirus penaei, or from the lymphoidorgans and hepatopancreas of uninfected shrimp. It is concluded that the DNA probe can specifically amplify DNA from RV-PJ and SEMBV.