Multiplex-PCR approach to identify Bacillus species applied in microbial fertilizers

Abstract:

[Objective] To discriminate the strains of Bacillus subtilis group including B. subtilis, B. amyloliquefaciens, B. licheniformis, and B. pumilus, a rapid and accurate distinguishing method is essential for the identification of the target strains to ensure the quality and safety of microbial fertilizers. [Methods] By analyzing unique nucleotide sequences of the rpoA, gyrA and 16S rDNA genes, 4 pairs of species-specific primers were optimized and the multiplex PCR was de-veloped to discriminate and identify B. subtilis, B. amyloliquefaciens, B. licheniformis and B. pumilus. [Results] Thirty-three reference strains belonging to three genera of Bacillus, Paenibacillus and Brevibacillus were tested and the anticipated results appeared except for four species with cross amplification results with the primers of B. pumilus. How-ever, the four species can be easily discriminated by morphology characters. In addition, the multiplex-PCR results of 23 strains of B. subtilis group isolated from MF products were identical with the biochemical assay. [Conclusion] The newly constructed multiplex-PCR assay is species-specific and effective. This method can be used to detect and identify the strains of B.subtilis group from microbial fertilizers products.

Key words:multiplex-PCR; species-specific primers; identification; Bacillus subtilis; microbial fertilizers