Screening of Bacillus thuringiensis serotypes by polymerase chain reaction (PCR) for insecticidal crystal genes toxic against coffee berry borer

Using PCR,257 isolates of Bacillus thuringiensis(Bt) were screened for cry-type genes. Of 257 isolates/strains, 60 isolates were identified as cry7/8, 10 isolates as cry3 and 36 isolates as cry 1I. One specific strain of B. thuringiensis (sumiyoshiensis; T03B 001) was investigated for the presence of cry7 and cry8 genes. Genes Cry7 and cry8 were first detected in this strain using family primers prior to analysis by exclusion polymerase chain reaction (E-PCR) using specific type primers. E-PCR conducted with the above said primers led to the identification by agarose gel electrophoresis of a remaining 1.5 Kb family band indicating a potentially novel gene. This PCR product, (1.5 Kb), was purified from the gel and cloned in pGEM-T Easy vector. Twenty recombinant colonies bearing 1.5 Kb insert were identified and three randomly selected representatives of the group, clones 7, 8 and 10, were sequenced and compared to all cry7 and cry8 sequences available from Gene Bank. Alignments with available DNA and protein sequences showed that all these clones contained a gene related to cry8Aa1. Analysis using protein sequence alignment showed that the sequence from clone 7 differed from the closest relative, known under the new nomenclature as cry 8Aa1, by 44%. The crystal proteins from B. thuringiensis sumiyoshiensis (T03B 001) was toxic to coffee berry borer larvae.