EFFECTIVE DETECTION OF BANANA BUNCHY TOP NANOVIRUS USING DNA PROBES

Abstract

According to DNA sequences of components one and four of BBTV isolate, that were
isolated from AGERI cultivated area, Egypt, two pairs of primers were designed to
amplify BBTV replicase (BBTV-rep) and movement protein (BBTV-mv) genes. The
amplified fragments were cloned into pGEM-T easy vector and two new plasmids
were created, i.e., pBBTV-Rep and BBTV-MV and transformed into Escherichia coli.
Results showed that PCR products with a size of about 1.1 Kbp, which represented
BBTV-rep and BBTV-mv, were successfully amplified. In case of BBTV-mv, a
number of 17 clones were subjected to minipreparation followed by restriction
endonuclease analysis. Results showed that 9 (1-5, 12-14 and 16) out of the 17 clones
were found to be recombinant and 1.1 Kbp fragments were released. For Southern
blotting analysis, the previous gel was southern blotted onto a membrane and the same
9 clones gave signals when the membrane was hybridized with 32P-labeled-PCR probe
of BBTV-mv amplified from DNA of BBTV-infected plants. On the other hand,
similar probe was prepared from pBBTV-MV plasmid and used for virus detection in
BBTV-infected plants. On the other hand, a PCR product amplified from the pBBTVRep
plasmid was labeled with 32P and used as a probe for detection of BBTV in virusinfected
plants as well as the healthy as control. Findings of this study could be
concluded in developing a rapid, effective and sensitive detection of BBTV in nucleic
acids of either PCR amplified from BBTV-infected plants or 32P-DNA-PCR labeled
probe via Southern blot hybridization