Efficiency of PCR-based methods in discriminating Bifidobacterium longum ssp. longum and Bifidobacterium longum ssp. infantis strains of human origin |
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Authors: | Srůtková Dagmar Spanova Alena Spano Miroslav Dráb Vladimír Schwarzer Martin Kozaková Hana Rittich Bohuslav |
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Affiliation: | a Department of Immunology and Gnotobiology, Institute of Microbiology ASCR, v.v.i., Doly 183, Nový Hrádek, 549 22, Czech Republicb Masaryk University, Faculty of Science, Institute of Experimental Biology, Tvrdého 14, Brno, 602 00, Czech Republicc Brno University of Technology, Faculty of Civil Engineering, Veve?í 331/95, Brno, 602 00, Czech Republicd MILCOM, Co. Ltd., Dairy Research Institute, 390 02 Tábor, Czech Republic |
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Abstract: | Bifidobacterium longum is considered to play an important role in health maintenance of the human gastrointestinal tract. Probiotic properties of bifidobacterial isolates are strictly strain-dependent and reliable methods for the identification and discrimination of this species at both subspecies and strain levels are thus required. Differentiation between B. longum ssp. longum and B. longum ssp. infantis is difficult due to high genomic similarities. In this study, four molecular-biological methods (species- and subspecies-specific PCRs, random amplified polymorphic DNA (RAPD) method using 5 primers, repetitive sequence-based (rep)-PCR with BOXA1R and (GTG)5 primers and amplified ribosomal DNA restriction analysis (ARDRA)) and biochemical analysis, were compared for the classification of 30 B. longum strains (28 isolates and 2 collection strains) on subspecies level. Strains originally isolated from the faeces of breast-fed healthy infants (25) and healthy adults (3) showed a high degree of genetic homogeneity by PCR with subspecies-specific primers and rep-PCR. When analysed by RAPD, the strains formed many separate clusters without any potential for subspecies discrimination. These methods together with arabionose/melezitose fermentation analysis clearly differentiated only the collection strains into B. longum ssp. longum and B. longum ssp. infantis at the subspecies level. On the other hand, ARDRA analysis differentiated the strains into the B. longum/infantis subspecies using the cleavage analysis of genus-specific amplicon with just one enzyme, Sau3AI. According to our results the majority of the strains belong to the B. longum ssp. infantis (75%). Therefore we suggest ARDRA using Sau3AI restriction enzyme as the first method of choice for distinguishing between B. longum ssp. longum and B. longum ssp. infantis. |
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Keywords: | PCR, Polymerase chain reaction RAPD, random amplified polymorphic DNA rep-PCR, repetitive sequence-based PCR ARDRA, amplified ribosomal DNA restriction analysis CCM, Czech collection of microorganisms ATCC, American Type and Culture Collection DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen MRS agar, de Man, Rogosa and Sharpe agar |
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