[Use of DIR-PCR for elaboration of molecular markers of intraspecies bacterial groups as exemplified by Bacillus thuringiensis].

ABSTRACT

A recently developed PCR-fingerprinting method, the so-called DIR (diverged inverted repeats)-PCR, was used for quick search for molecular markers of Bacillus thuringiensis subspecies carrying the cry1 genes. The analysis of the fingerprints obtained by this method made it possible to reveal PCR fragments characteristic of the subspecies that produce proteins toxic for insects of the order Lepidoptera. Cloning and sequencing of these fragments allowed systems of SCAR (sequence characterized amplified region) primers to be designed, which are specific to the above group of B. thuringiensis strains. Comparison of the specific fragments with sequences available in the GenBank database revealed their homology with the rpoC gene family and the adjacent spacer region, suggesting chromosomal localization of these markers. This increases the reliability of the designed system of SCAR primers, because plasmids may be lost or transferred by transformation between closely related strains. It was demonstrated that the DIR-PCR method allows markers to be developed that are linked to diagnostic genotypic and phenotypic characteristics of bacteria.