Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains

An extended PCR method was established to rapidly identify and classify Bac illus thuringiensis strains containing cry (crystal protein) genes toxic to lepidopteran, coleopteran, and dipteran pests (Ben-Dov et al., Appl. Envir on. Microbiol. 63:4883-4890, 1997). To optimize identification of all repor ted cry genes, this methodology needs a complete PCR set of primers. In the study reported here, a set of universal (Un9) and specific primers for mul tiplex rapid screening for all four known genes from the cry9 group was des igned. PCR analyses were performed for cry9 genes on 16 standard strains an d 215 field isolates of B. thuringiensis, Among the standard strains, only B. thuringiensis subsp. aizawai RD-133, which harbors cry1 and cry2 genes, was positive with Un9 but negative to all four specific primers for cry9 ge nes. DNA of 22 field-collected isolates was also found to be positive with Un9. These isolates were classified into three cry9 profiles using specific primers; all of them harbor cry1 and cry2. This newly designed set of prim ers complements the existing PCR methodology for most currently form cry ge nes.