African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5173

Full Length Research Paper

Use of novel specific primers targeted to pheS and tuf gene for species and subspecies identification and differentiation of the Bacillus subtilis subsp. subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis

Chien-Hsun Huang*
  • Chien-Hsun Huang*
  • Bioresource Collection and Research Center, Food Industry Research and Development Institute, 331 Shih-Pin Road, Hsinchu 30062, Taiwan, ROC
  • Google Scholar
Lina Huang
  • Lina Huang
  • Bioresource Collection and Research Center, Food Industry Research and Development Institute, 331 Shih-Pin Road, Hsinchu 30062, Taiwan, ROC
  • Google Scholar
Mu-Tzu Chang
  • Mu-Tzu Chang
  • Department of Animal Science, National Chiayi University, 300 Syuefu Rd., Chiayi City 60004, Taiwan.
  • Google Scholar
Chean-Pin Wu
  • Chean-Pin Wu
  • Department of Animal Science, National Chiayi University, 300 Syuefu Rd., Chiayi City 60004, Taiwan.
  • Google Scholar


  •  Received: 03 January 2017
  •  Accepted: 26 January 2017
  •  Published: 21 February 2017

Abstract

To clearly delimit the members of the Bacillus subtilis group (BSG), is difficult using common phenotypic and genotypic methods. This study described the use of pheS and tuf gene as targets for interspecies discrimination within the BSG, and also to develop specific PCR and SNP primers for species and subspecies identification and differentiation. The average sequence similarity values of the pheS and tuf gene among type strains were 85.1 and 94.7%, respectively, and all members of the BSG could be clearly distinguished based on phylogenetic analyses of pheS gene sequence. In addition, the specific primers were designed according to pheS and tuf gene sequence. The primers were shown to specifically identify B. subtilis subsp. subtilis, B. amyloliquefaciens and Bacillus licheniformis, and clearly differentiate the subspecies of B. amyloliquefaciens using specific-PCR, combined with two-plex minisequencing method. In conclusion, we have successfully established a comparative sequence analysis and rapid molecular diagnosis techniques for determination of interspecies within the BSG.

Key words: Bacillus subtilis group (BSG), species and subspecies discrimination, comparative sequence analysis, specific Pcr, two-plex minisequencing.