DOI QR코드

DOI QR Code

Quantitative Real-time PCR using Lactobacilli as Livestock Probiotics

Real-time PCR을 이용한 가축생균제용 유산균 정량분석

  • Choi, Yeon-Jae (Department of Animal Science & Technology, Sunchon National University) ;
  • Kim, Sun-Ho (Department of Animal Science & Technology, Sunchon National University) ;
  • Gu, Min-Jeong (Department of Animal Science & Technology, Sunchon National University) ;
  • Choe, Han-Na (Depart of Biology, Sunchon National University) ;
  • Kim, Dong-Un (National Institute of Animal Science, RDA) ;
  • Cho, Sang-Bum (Division of Animal Life Science, Konkuk University) ;
  • Kim, Su-Ki (Division of Animal Life Science, Konkuk University) ;
  • Jeon, Che-Ok (Department of Life Sciences, ChungAng University) ;
  • Bae, Gui-Seok (Department of Animal Science & Technology, ChungAng University) ;
  • Lee, Sang-Seok (Department of Animal Science & Technology, Sunchon National University)
  • 최연재 (순천대학교 동물자원과학과) ;
  • 김선호 (순천대학교 동물자원과학과) ;
  • 구민정 (순천대학교 동물자원과학과) ;
  • 최한나 (순천대학교 생물학과) ;
  • 김동운 (농촌진흥청 국립축산과학원) ;
  • 조상범 (건국대학교 동물생명과학부) ;
  • 김수기 (건국대학교 동물생명과학부) ;
  • 전체옥 (중앙대학교 생명과학과) ;
  • 배귀석 (중앙대학교 동물자원과학과) ;
  • 이상석 (순천대학교 동물자원과학과)
  • Received : 2010.11.20
  • Accepted : 2010.11.23
  • Published : 2010.12.30

Abstract

This study was conducted using quantitative real-time PCR using Lactobacilli as probiotics. Quantitative real-time PCR (RT PCR) was conducted via a method involving SYBR Green 1 and a probe. Plasmid DNA was cloned using the 16S-23S rRNA intergenic species region. Gene clones were diluted from $10^2$ to $10^{10}$. Standard curves were constructed via Ct values obtained from the results of Real-time PCR via the aforementioned SYBR Green 1 and probe method. Plasmid DNA was also cloned using the 16S-23S rRNA intergenic species region and the gene clones were diluted from $10^2$ to $10^{10}$ copy numbers via the probe method. Using RT PCR, a standard curve of plasmid DNA copy numbers was also determined. The slope value for the Y-axis intercept and $R^2$ value were measured as -3.346, 33.18, and 0.993, respectively, via the first method. For the second method, the slope value for the Y-axis intercept and $R^2$ were -3.321, 31.10 and 0.995, respectively. The PCR inhibitor could not express the detection curve at a copy number over $10^{10}$ via either method, owing to high DNA density. The DNA extract from probiotics was diluted without pre-culturing, and 16 products were amplified via both methods. The Ct value was 11.06~18.12 in the first method and 16.74~22.11 in the second method. Measured probiotics and log copy values were largely similar among the methods used. It was concluded that both methods are effective for analysis, but further research will be required to verify the optimal method.

Acknowledgement

Supported by : 농촌진흥청

References

  1. Annuk, H, J. Shchepetova, T. Kullisaar, E. Songisepp, M. Zilmer, and M. Mikelssar. 2003. Characterization of intestinal Lactobacillus as probiotics candidates. J. Appl. Microbiol. 94, 403-412. https://doi.org/10.1046/j.1365-2672.2003.01847.x
  2. Berg, R. D. 1998. Probiotics, prebiotics or conbiotics. Trends Microbiol. 6, 89-92. https://doi.org/10.1016/S0966-842X(98)01224-4
  3. Bohaychuk, V. M., G. E. Gensler, M. E. McFall, R. K. King, and D. G. Renter. 2007. A real-time PCR assay for the detection of Salmonella in a wide variety of food and food animal matrices. J. Food Protect. 70, 1080-1087.
  4. Catarame, T. M. G., K. A. O’Hanlon, D. A. McDowell, I. S. Blair, and G. Duffy. 2006. Comparison of a real-time polymerase chain reaction assay with a culture method for the detection of Salmonella in retail meat samples. J. Food Safety 26, 1-15.
  5. Cheung, P. Y., K. K. Kwok, and K. M. Kam. 2007. Application of BAX system, Tecra Unique TM Salmonella test, and a conventional culture method for the detection of Salmonella in ready-to-eat and raw foods. J. Appl. Microbiol. 103, 219-227. https://doi.org/10.1111/j.1365-2672.2006.03210.x
  6. Choi, J. P. 2005. Quantitative analysis by Real-time PCR and Physiological properties of Food-borne Pathogens. Kyeong-won univ. 55.
  7. Conway, P. L., S. L. Gorbach, and B. R. Coldin. 1987. Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells. J. Daily Sci. 70, 1-12. https://doi.org/10.3168/jds.S0022-0302(87)79974-3
  8. Dubernet, S., N. Desmasures, and M. Gueguen. 2002. A PCR-based method for identification of Lactobacilli at genus level. FEMS Microbiol. Lett. 214, 271. https://doi.org/10.1111/j.1574-6968.2002.tb11358.x
  9. Han, S. R., J. Y. Hyeon, H. Y. Kim, J. S. Park, H. S. Shin, and K. H. Seo. 2008. Evaluation of conventional culture methods and validation of immunoassays for rapid detection of Listeria monocytogenes in daily and processed foods. Korean J. Food Sci. Ani. Resour. 286, 16-622. https://doi.org/10.5851/kosfa.2008.28.5.616
  10. Hein, I., G. Flekna, M. Krassnig, and M. Wagner. 2006. Real-time PCR for the detection of Salmonella spp. In food: An alternative approach to a conventional PCR system suggested by the Food-PCR project. J. Microbiol. Methods 66, 538-547. https://doi.org/10.1016/j.mimet.2006.02.008
  11. Higuchi, R., C. Fockler, G. Dollinger, and R. Waston. 1993. Kinetic PCR: Real monitoring of DNA amplication reactions. Biotechnology 11, 1026. https://doi.org/10.1038/nbt0993-1026
  12. Kim, Y. Y. Study for nutrition diarrhea prevention in weaning pigs by controlling intestinal microflora. Ministry for food, agriculture, forestry and fisheries. Research paper. 38.
  13. Korean Feed Processing. Subsidiary feeder guideline. Attach documents 3.
  14. Krascsenicsova, K., L. Piknova, E. Kaclikova, and T. Kuchta. 2008. Detection of Salmonella enteric in food using two-step enrichment and real-time PCR. Lett. Appl. Microbiol. 46, 483-487. https://doi.org/10.1111/j.1472-765X.2008.02342.x
  15. Lee, E. Y. 2008. Problems and verification system of probiotics as livestock-enviroment improving agent produced. Korean J. Microbiol. Biotechnol. 36, 87-95.
  16. Lee, T. S., B. K. Park, and D. H. Oh. 2003. Detection of pathogenic Yersinia Enterocolitica in drinking water and vegetables by multiplex PCR. J. Korean Soc. Food Sci. Nutr. 32, 35-41 https://doi.org/10.3746/jkfn.2003.32.1.035
  17. Malorny, B., E. Paccassoni, P. Fach, C. Bunge, A. Martin, and R. Helmuth. 2004. Diagnostic real-time PCR for detection of Salmonella in food. Appl. Environ. Microbiol. 70, 7046-7052. https://doi.org/10.1128/AEM.70.12.7046-7052.2004
  18. Monique, H. and K. Jan. 2006. Quantitative Real-time PCR analysis of fecal Lactobacillus species in infants receiving a prebiotic infant formula. Appl. Environ. Microbiol. 72, 2359-2365. https://doi.org/10.1128/AEM.72.4.2359-2365.2006
  19. Paik, H. D., M. Y. Jung, H. Y. Jung, W. S. Kim, and K. T. Kim. 2002. Characterization of Bacillus polyfermenticus SCD for oral Bacteriotherapy of Gastrointestinal Disorders. Korean J. Food Sci. Technol. 34, 73-78.
  20. Palomares, C., M. J. Torres, A. Torres, J. Aznar, and J. C. Palomares. 2003. Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR. Diagn. Microbiol. Infect. Dis. 45, 183-189. https://doi.org/10.1016/S0732-8893(02)00542-4
  21. Uyttendaele, M., K. Vanwildemeersch, and J. Debevere. 2003. Evaluation of real-time PCR vs automated ELISA and a conventional culture method using a semi-solid medium for detection of salmonella. Lett. Appl. Microbiol. 37, 386-391. https://doi.org/10.1046/j.1472-765X.2003.01415.x
  22. Yeh, K. S., C. E. Tsai, S. P. Chen, and C. W. Liao. 2002. Comparison between VIDAS automatic enzyme-linked fluorescent immunoassay and culture method for Salmonella recovery from pork carcass sponge samples. J. Food Protect 65, 1656-1659.

Cited by

  1. A Study Bioremediation of Tidal Flat by Microorganism in Pilot Scale Test vol.24, pp.10, 2014, https://doi.org/10.5352/JLS.2014.24.10.1110