title: Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA, rpoB Gene Sequencing and RAPD-PCR.
abstract: Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.
authors.affiliation: Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran.|Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
authors.forename: Milad
authors.initials: M
authors.lastname: Mohkam
authors.affiliation: Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran.
authors.forename: Navid
authors.initials: N
authors.lastname: Nezafat
authors.affiliation: Faculty of Science and Engineering, School of Engineering, University of Waikato, Private Bag 3105, Hamilton, 3240, New Zealand.
authors.forename: Aydin
authors.initials: A
authors.lastname: Berenjian
authors.affiliation: Noncommunicable Diseases Research Center, Fasa University of Medical Sciences, Fasa, Iran.|Department of Medical Biotechnology, School of Medicine, Fasa University of Medical Sciences, Fasa, Iran.
authors.forename: Mohammad Ali
authors.initials: MA
authors.lastname: Mobasher
authors.affiliation: Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran. ghasemiy@sums.ac.ir.|Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran. ghasemiy@sums.ac.ir.|Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran. ghasemiy@sums.ac.ir.
authors.forename: Younes
authors.initials: Y
authors.lastname: Ghasemi
mesh_terms: D001407:Bacillus / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005798:Genes, Bacterial; D019936:Probiotics; D012329:RNA, Bacterial / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D019105:Random Amplified Polymorphic DNA Technique; D011938:Rec A Recombinases / Q000235:genetics; D058441:Rhizosphere; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013045:Species Specificity
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D011938:Rec A Recombinases; D012321:DNA-Directed RNA Polymerases
keywords: Bacillus; Concatenation; Multilocus sequence analysis (MLSA); Probiotics; Randomly amplified polymorphic DNA-PCR (RAPD-PCR); recA; rpoB