Journal of Microbiology and Biotechnology

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

DOI QR Code

Development of Real-Time PCR for the Detection of Clostridium perfringens in Meats and Vegetables

  • Chon, Jung-Whan (KU Center for Food Safety, Veterinary Science Research Institute and College of Veterinary Medicine, Konkuk University) ;
  • Park, Jong-Seok (Division of Research Planning and Management, Korea Food and Drug Administration) ;
  • Hyeon, Ji-Yeon (KU Center for Food Safety, Veterinary Science Research Institute and College of Veterinary Medicine, Konkuk University) ;
  • Park, Chan-Kyu (Department of Animal Biotechnology, College of Animal Bioscience and Technology, Konkuk University) ;
  • Song, Kwang-Young (KU Center for Food Safety, Veterinary Science Research Institute and College of Veterinary Medicine, Konkuk University) ;
  • Hong, Kwang-Won (Department of Food Science and Technology, Dongguk University) ;
  • Hwang, In-Gyun (Division of Microbiology, Korea Food and Drug Administration) ;
  • Kwak, Hyo-Sun (Division of Foodborne Diseases Prevention and Surveillance, Korea Food and Drug Administration) ;
  • Seo, Kun-Ho (KU Center for Food Safety, Veterinary Science Research Institute and College of Veterinary Medicine, Konkuk University)
  • Received : 2011.07.29
  • Accepted : 2011.12.07
  • Published : 2012.04.28
Download PDF
⟨ Previous Next ⟩

Abstract

A real-time PCR assay was developed and validated inhouse specifically for the detection of Clostridium perfringens (Cl. perfringens) in meats and vegetables by comparing with the culture method. The detection limit of the real-time PCR assay in phosphate-buffered saline was $10^2$ CFU/ml. When the two methods were compared in food samples inoculated with Cl. perfringens, the culture method detected 52 positives, whereas real-time PCR detected 51 positives out of 160 samples. The difference was without statistical significance (p>0.05). Real-time PCR assay is an option for quality assurance laboratories to perform standard diagnostic tests, considering its detection ability and time-saving efficiency.

Keywords

References

  1. Albini, S., I. Brodard, A. Jaussi, N. Wollschlaeger, J. Frey, R. Miserez, and C. Abril, 2008. Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates. Vet. Microbiol. 127: 179-185. https://doi.org/10.1016/j.vetmic.2007.07.024
  2. Brynestad, S. and P. E. Granum. 2002. Clostridium perfringens and food borne infections. Int. J. Food Microbiol. 74: 195-202. https://doi.org/10.1016/S0168-1605(01)00680-8
  3. Cai, T., L. Jiang, C. Yang, and K. Huang. 2006. Application of real-time PCR for quantitative detection of Vibrio parahaemolyticus from seafood in eastern China. FEMS Immunol. Med. Microbiol. 46: 180-186. https://doi.org/10.1111/j.1574-695X.2005.00016.x
  4. Chon, J. H., J. Y. Hyeon, I. G. Hwang, H. S. Kwak, J. A. Han, Y. H. Chung, et al. 2010. Comparison of standard culture method and real-time PCR for detection of Vibrio parahaemolyticus in seafoods and vegetables. Korean J. Food Sci. Technol. 42: 355-360.
  5. Erol, I., M. Goncuoglu, N. D. Ayaz, F. S. Bilir-Ormanci, and G. Hildebrandt. 2008. Molecular typing of Clostridium perfringens isolated from turkey meat by multiplex PCR. Lett. Appl. Microbiol. 47: 31-34. https://doi.org/10.1111/j.1472-765X.2008.02379.x
  6. Fukushima, H., K. Katsube, Y. Hata, R. Kishi, and S. Fujiwara. 2007. Rapid separation and concentration of food-borne pathogens in food samples prior to quantification by viable-cell counting and real-time PCR. Appl. Environ. Microbiol. 73: 92-100. https://doi.org/10.1128/AEM.01772-06
  7. Gurjar, A. A., N. V. Hegde, B. C. Love, and B. M. Jayarao. 2008. Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces of dairy cattle. Mol. Cell. Probes 22: 90-95. https://doi.org/10.1016/j.mcp.2007.08.001
  8. Hyeon, J. Y., I. G. Hwang, H. S. Kawk, J. S. Park, S. Heo, I. S. Choi, et al. 2009. Evaluation of an automated ELISA and real-time PCR by comparing with a conventional culture method for the detection of Salmonella spp. in steamed pork and raw broccoli sprouts. Korean J. Food Sci. Anim. Resour. 29: 506-512. https://doi.org/10.5851/kosfa.2009.29.4.506
  9. Jung, S. H., M. J. Hur, J. H. Ju, K. A. Kim, S. S. Oh, J. M. Go, et al. 2006. Microbiological evaluation of raw vegetables. J. Food Hyg. Safety 21: 250-257.
  10. Lantz, P. G., R. Knutsson, Y. Blixt, W. A. Al-Soud, E. Borch, and P. Radstrom. 1998. Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR, a study of sample preparation and PCR-inhibitory components. Int. J. Food Microbiol. 45: 93-105. https://doi.org/10.1016/S0168-1605(98)00152-4
  11. Lee, J. H., K. Y. Song, J. Y. Hyeon, I. G. Hwang, H. S. Kwak, J. A. Han, et al. 2010. Comparison of standard culture method and real-time PCR assay for detection of Staphylococcus aureus in processed and unprocessed foods. Korean J. Food Sci. Anim. Resour. 30: 410-418. https://doi.org/10.5851/kosfa.2010.30.3.410
  12. 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
  13. Petit, L., M. Gibert, and M. R. Popoff. 1999. Clostridium perfringens: Toxinotype and genotype. Trends Microbiol. 7: 104-110. https://doi.org/10.1016/S0966-842X(98)01430-9
  14. Sartory, D. P., M. Field, S. M. Curbishley, and A. M. Pritchard. 1998. Evaluation of two media for the membrane filtration enumeration of Clostridium perfringens from water. Lett. Appl. Microbiol. 27: 323-327. https://doi.org/10.1046/j.1472-765X.1998.00454.x
  15. Seo, K. H. and R. E. Brackett. 2005 Rapid, specific detection of Enterobacter sakazakii in infant formula using a real-time PCR assay. J. Food Prot. 68: 59-63. https://doi.org/10.4315/0362-028X-68.1.59
  16. Wu, S. B., N. Rodgers, and M. Choct. 2011. Real-time PCR assay for Clostridium perfringens in broiler chickens in a challenge model of necrotic enteritis. Appl. Environ. Microbiol. 77: 1135-1139. https://doi.org/10.1128/AEM.01803-10

Cited by

  1. Rapid, Sensitive, and Specific Detection of Clostridium tetani by Loop-Mediated Isothermal Amplification Assay vol.23, pp.1, 2012, https://doi.org/10.4014/jmb.1205.05063
  2. 유제품과 육제품에서 황색포도상구균 신속검출을 위한 PCR법의 비교검증 vol.45, pp.6, 2012, https://doi.org/10.9721/kjfst.2013.45.6.791
  3. Molecular Epidemiology ofClostridium perfringensIsolated from Food Poisoning in Seoul, 2013 vol.44, pp.2, 2014, https://doi.org/10.4167/jbv.2014.44.2.170
  4. Comparison of Culture, Conventional and Real-time PCR Methods for Listeria monocytogenes in Foods vol.34, pp.5, 2014, https://doi.org/10.5851/kosfa.2014.34.5.665
  5. Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes vol.15, pp.None, 2012, https://doi.org/10.1186/s12866-015-0561-y
  6. Rapid Detection of Lactobacillus kefiranofaciens in Kefir Grain and Kefir Milk Using Newly Developed Real-Time PCR vol.78, pp.4, 2012, https://doi.org/10.4315/0362-028x.jfp-14-329
  7. Prevalence and toxin type of Clostridium perfringens in beef from four different types of meat markets in Seoul, Korea vol.26, pp.2, 2012, https://doi.org/10.1007/s10068-017-0075-5
  8. Prevalence, toxin gene profile, antibiotic resistance, and molecular characterization of Clostridium perfringens from diarrheic and non-diarrheic dogs in Korea vol.19, pp.3, 2018, https://doi.org/10.4142/jvs.2018.19.3.368
  9. Detection of pathogenic microorganisms from bloodstream infection specimens using TaqMan array card technology vol.8, pp.None, 2012, https://doi.org/10.1038/s41598-018-31200-3
  10. Large-Scale Genomic Analyses and Toxinotyping of Clostridium perfringens Implicated in Foodborne Outbreaks in France vol.10, pp.None, 2012, https://doi.org/10.3389/fmicb.2019.00777
  11. Effect of Saccharomyces boulardii Supplementation on Performance and Physiological Traits of Holstein Calves under Heat Stress Conditions vol.9, pp.8, 2012, https://doi.org/10.3390/ani9080510
  12. In Situ Processing and Efficient Environmental Detection (iSPEED) of tree pests and pathogens using point-of-use real-time PCR vol.15, pp.4, 2020, https://doi.org/10.1371/journal.pone.0226863
  13. Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies vol.13, pp.4, 2012, https://doi.org/10.3390/toxins13040266
  14. A novel in situ methodology for visual detection of Clostridium perfringens in pork harnessing saltatory rolling circle amplification vol.69, pp.None, 2012, https://doi.org/10.1016/j.anaerobe.2021.102324
  15. Clostridium Perfringens Toxin Types Associated with Meat: Review in Iran vol.15, pp.4, 2012, https://doi.org/10.30699/ijmm.15.4.384