Multiplex-PCR Approach to Identify Bacillus megaterium and Bacillus cereus Group Applied in Microbial Fertilizers

Abstract:

Bacillus megaterium strains are commonly used in microbial fertilizer (MF). MF products are often contaminated by other B. cereus group members, which have similar phenotype such as Bacillus cereus, B. thuringiensis, B. mycoide. For quality control and safety of MF, a rapid and accurate method is needed to distinguish the strains of Bacillus megaterium from B. cereus group. Based on specific nucleotide sequences of the spoOA genes, 2 pairs of species-specific primers were designed and a multiplex-PCR (mPCR) was developed for this purpose. When the optimized mPCR was used to detect the DNAs of 24 reference strains from three genera of Bacillus, Paenibacillus, and Brevibacillus, all B. megaterium strains showed single fragment of 443 bp and Bacillus cereus group showed a fragment of 411 bp. However, no any amplified product was from the other bacteria. The sensitivity of mPCR was 105 CFU/mL. The mPCR results of 10 isolates of B. megaterium/B. cereus group and 8 products of MF coincided with the biochemical assay. Taken together, our newly developed mPCR assay was species-specific and effective in application. It can be used to detect and identify the strains of B. megaterium and B. cereus group from microbial fertilizer products.