Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Development of Strain-Specific Primers for Identification of Bifidobacterium bifidum BGN4

  • Youn, So Youn (Animal and Plant Quarantine Agency) ;
  • Ji, Geun Eog (Department of Food and Nutrition, Research Institute of Human Ecology, Seoul National University) ;
  • Han, Yoo Ri (Department of Food and Nutrition, Research Institute of Human Ecology, Seoul National University) ;
  • Park, Myeong Soo (Research Institute, Bifido Co., Ltd.)
  • Received : 2016.10.10
  • Accepted : 2017.02.24
  • Published : 2017.05.28
Download PDF
⟨ Previous Next ⟩

Abstract

Bifidobacterium bifidum BGN4 (BGN4) has many proven beneficial effects, including antiallergy and anticancer properties. It has been commercialized and used in several probiotic products, and thus strain-specific identification of this strain is very valuable for further strain-dependent physiological study. For this purpose, we developed novel multiplex polymerase chain reaction (PCR) primer sets for strain-specific detection of BGN4 in commercial products and fecal samples of animal models. The primer set was tested on seven strains of B. bifidum and 75 strains of the other Bifidobacterium species. The BGN4-specific regions were derived using megaBLAST against genome sequences of various B. bifidum databases and four sets of primers were designed. As a result, only BGN4 produced four PCR products simultaneously whereas the other strains did not. The PCR detection limit using BGN4-specific primer sets was $2.8{\times}10^1CFU/ml$ of BGN4. Those primer sets also detected and identified BGN4 in the probiotic products containing BNG4 and fecal samples from a BGN4-fed animal model with high specificity. Our results indicate that the PCR assay from this study is an efficient tool for the simple, rapid, and reliable identification of BGN4, for which probiotic strains are known.

Keywords

References

  1. Arboleya S, Ruas-Madiedo P, Margolles A, Solis G, Salminen S, de Los Reyes-Gavilan CG, et al. 2010. Characterization and in vitro properties of potentially probiotic Bifidobacterium strains isolated from breast-milk. Int. J. Food Microbiol. 149: 28-36.
  2. Frick JS, Schenk K, Quitadamo M, Kahl F, Koberle M, Bohn E, et al. 2007. Lactobacillus fermentum attenuates the proinflammatory effect of Yersinia enterocolitica on human epithelial cells. Inflamm. Bowel Dis. 13: 83-90. https://doi.org/10.1002/ibd.20009
  3. Guarner F, Malagelada JR. 2003. Bacterial flora of the digestive tract. J. Gastroenterol. Hepatol. 26Suppl: 11-15.
  4. Macpherson AJ, Harris NL. 2004. Interactions between commensal intestinal bacteria and the immune system. Nat. Rev. Immunol. 4: 478-485. https://doi.org/10.1038/nri1373
  5. Picard C, Fioramonti J, Francois A, Robinson T, Neant F, Matuchansky C. 2005. Bifidobacteria as probiotic agents - physiological effects and clinical benefits. Aliment. Pharmacol. Ther. 22: 495-512. https://doi.org/10.1111/j.1365-2036.2005.02615.x
  6. Venema K, Maathuis AH. 2003. A PCR-based method for identification of bifidobacteria from the human alimentary tract at the species level. FEMS Microbiol. Lett. 224: 143-149. https://doi.org/10.1016/S0378-1097(03)00436-1
  7. Gill H, Prasad J. 2008. Probiotics, immunomodulation, and health benefits. Adv. Exp. Med. Biol. 606: 423-454.
  8. Lin DC. 2003. Probiotics as functional foods. Nutr. Clin. Pract. 18: 497-506. https://doi.org/10.1177/0115426503018006497
  9. You HJ, Oh DK, Ji GE. 2004. Anticancerogenic effect of a novel chiroinositol-containing polysaccharide from Bifidobacterium bifidum BGN4. FEMS Microbiol. Lett. 240: 131-136. https://doi.org/10.1016/j.femsle.2004.09.020
  10. Kim IH, Park MS, Ji GE. 2003. Characterization of adhesion of Bifidobacterium sp. BGN4 to human enterocyte-like Caco-2 cells. J. Microbiol. Biotechnol. 13: 276-281.
  11. Kim H, Kwack K, Kim DY, Ji GE. 2005. Oral probiotic bacterial administration suppressed allergic responses in an ovalbumin-induced allergy mouse model. FEMS Immunol. Med. Microbiol. 45: 259-267. https://doi.org/10.1016/j.femsim.2005.05.005
  12. Kim JY, Kwon JH, Ahn SH, Lee SI, Han YS, Choi YO, et al. 2010. Effect of probiotic mix (Bifidobacterium bifidum, Bifidobacterium lactis, Lactobacillus acidophilus) in the primary prevention of eczema: a double-blind, randomized, placebocontrolled trial. Pediatr. Allergy Immunol. 21: e386-e393. https://doi.org/10.1111/j.1399-3038.2009.00958.x
  13. Kim NJ, Park SJ, Yum EM, Kim HY, Lee SH, Min JH, et al. 2003. Effect of Bifidobacterium-fermented soy hypocotyls intake on the composition of human large intestinal bacteria in the elderly. Food Sci. Biotechnol. 24: 2023-2026.
  14. Coudeyras S, Marchandin H, Fajon C, Forestier C. 2008. Taxonomic and strain-specific identification of the probiotic strain Lactobacillus rhamnosus 35 within the Lactobacillus casei group. Appl. Environ. Microbiol. 74: 2679-2689. https://doi.org/10.1128/AEM.02286-07
  15. Yeung PS, Kitts C L, Cano R, Tong PS, Sanders ME. 2004. Application of genotypic and phenotypic analyses to commercial probiotic strain identity and relatedness. J. Appl. Microbiol. 97: 1095-1104. https://doi.org/10.1111/j.1365-2672.2004.02400.x
  16. Yuki N, Watanabe K, Mike A, Tagami Y, Tanaka R, Ohwaki M, et al. 1999. Survival of a probiotic, Lactobacillus casei strain Shirota, in the gastrointestinal tract: selective isolation from faeces and identification using monoclonal antibodies. Int. J. Food Microbiol. 48: 51-57. https://doi.org/10.1016/S0168-1605(99)00029-X
  17. Fujimoto J, Tanigawa K, Kudo Y, Makino H, Watanabe K. 2011. Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide. J. Appl. Microbiol. 110: 209-217. https://doi.org/10.1111/j.1365-2672.2010.04873.x
  18. Maruo T, Sakamoto M, Toda T, Benno Y. 2006. Monitoring the cell number of Lactococcus lactis subsp. cremoris FC in human feces by real-time PCR with strain-specific primers designed using the RAPD technique. Int. J. Food Microbiol. 110: 69-76. https://doi.org/10.1016/j.ijfoodmicro.2006.01.037
  19. Sisto A, De Bellis P, Visconti A, Morelli L, Lavermicocca P. 2009. Development of a PCR assay for the strain-specific identification of probiotic strain Lactobacillus paracasei IMPC2.1. Int. J. Food Microbiol. 136: 59-65. https://doi.org/10.1016/j.ijfoodmicro.2009.09.017
  20. Endo A, Aakko J , Salminen S. 2012. Evaluation of s trainspecific primers for identification of Lactobacillus rhamnosus GG. FEMS Microbiol. Lett. 337: 120-125. https://doi.org/10.1111/1574-6968.12015
  21. Toshimitsu T, Nakamura M, Ikegami S, Terahara M, Itou H. 2013. Strain-specific identification of Bifidobacterium bifidum OLB6378 by PCR. Biosci. Biotechnol. Biochem. 77: 572-576. https://doi.org/10.1271/bbb.120803
  22. Germond JE, Mamin O, Mollet B. 2002. Species specific identification of nine human Bifidobacterium spp. in feces. Syst. Appl. Microbiol. 25: 536-543. https://doi.org/10.1078/07232020260517670
  23. Kwon HS, Yang EH, Lee SH, Yeon SW, Kang BH, Kim TY. 2005. Rapid identification of potentially probiotic Bifidobacterium species by multiplex PCR using speciesspecific primers based on the region extending from 16S rRNA through 23S rRNA. FEMS Microbiol. Lett. 250: 55-62. https://doi.org/10.1016/j.femsle.2005.06.041
  24. Matsuki T, Watanabe K, Tanaka R. 2003. Genus- and species-specific PCR primers for the detection and identification of bifidobacteria. Curr. Issues Intest. Microbiol. 4: 61-69.
  25. Yu DS, Jeong H, Lee DH, Kwon SK, Song JY, Kim BK, et al. 2012. Complete genome sequence of the probiotic bacterium Bifidobacterium bifidum strain BGN4. J. Bacteriol. 194: 4757-4758. https://doi.org/10.1128/JB.00988-12
  26. Kim JY, Choi YO, Ji GE. 2008. Effect of oral probiotics (Bifidobacterium lactis AD011 and Lactobacillus acidophilus AD031) administration on ovalbumin-induced food allergy mouse model. J. Microbiol. Biotechnol. 18: 1393-1400.
  27. Matsuki T, Watanabe K, Fujimoto J, Miyamoto Y, Takada T, Matsumoto K, et al. Development of 16S rRNA-genetargeted group-specific primers for the detection and identification of predominant bacteria in human feces. Appl. Environ. Microbiol. 68: 5445-5451.

Cited by

  1. Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96 well plate vol.20, pp.None, 2017, https://doi.org/10.1186/s12866-020-01781-z
  2. Deoxycholic Acid Upregulates Serum Golgi Protein 73 through Activating NF-κB Pathway and Destroying Golgi Structure in Liver Disease vol.11, pp.2, 2017, https://doi.org/10.3390/biom11020205