Browse > Article
http://dx.doi.org/10.13103/JFHS.2021.36.1.93

Establishment of Sample Preparation Method for PCR Detection of Clostridium perfringens from Agricultural Products  

Choi, Song-Yi (Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Administration)
Seo, Min-Kyoung (Food Safety Risk Assessment Division, Natioanal Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety)
Yoon, Jae-Hyun (Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Administration)
Rajalingam, Nagendran (Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Administration)
Hwang, Injun (Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Administration)
Kim, Se-Ri (Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Administration)
Publication Information
Journal of Food Hygiene and Safety / v.36, no.1, 2021 , pp. 93-99 More about this Journal
Abstract
This study was undertaken to compare the efficacy of different sample preparation (stomaching, pulsifying, and sonication) and DNA extraction methods (boiling and commercial kit) for detection of enterotoxin-producing Clostridium perfringens from produce by polymerase chain reaction (PCR). Each produce type was inoculated at concentrations of 102, 103, 104, 105, 106, and 107 spores/g. Produce inoculated with spores was treated with three sample preparation methods, and DNA was extracted by boiling method and a commercial kit, followed by PCR. The detection limit of stomached samples was lower than that of pummeled and sonicated samples by 10-100 times. Moreover, the DNA extraction efficiency of the commercial kit was found to be superior to that of boiling. In particular, the PCR efficiency of cherry tomato and perilla leaf samples was greatly affected by sample preparation and DNA extraction method. These data suggest that DNA extraction with a commercial kit after pulsification is an optimum sample preparation method for detection of C. perfringens by PCR.
Keywords
Clostridium perfringens; Sample preparation method; Produce; Polymerase chain reaction;
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
연도 인용수 순위
1 Petit, L., Gibert, M., Popoff, M.R., Clostridium perfringens toxinotype and genotype. Trends Microbiol., 7, 104-110 (1999).   DOI
2 Harrison, B., Raju, D., Garmory, H.S., Brett, M.M., Titball, R.W., Sarker, M.R., Molecular characterization of Clostridium perfringens isolates from humans with sporadic diarrhea: evidence for transcriptional regulation of the beta2-toxinencoding gene. Appl. Environ. Microbiol., 71, 8362- 8370 (2005).   DOI
3 Sparks, S.G., Carman, R.J., Sarker, M.R., McClane, B.A., Genotyping of enterotoxigenic Clostridium perfringens fecal isolates associated with antibiotic-associated diarrhea and food poisoning in North America. J. Clin. Microbiol., 39, 883-888 (2001).   DOI
4 Ministry of Food and Drug Safety, (2020, September 5). Statistics of food borne outbreaks. Retrieved from http://foodsafetykorea.go.kr/portal/healthyfoodlife/foodPoisoningStat
5 National Institute of Food and Drug Safety Evaluation, 2018. Chaper 2. Risk assessment, risk assessment of Clostridium perfringens in red pepper powder, shredded red pepper and composite seasonings. Cheongju, Korea. pp. 5-15.
6 Jung, S.H., Hur, M.J., Ju, H.H, Kim, J.A., Oh, S.S., Go, J.M., Kim, Y.H., Im, J., Microbiological evaluation of raw vegetables. J. Food Hyg. Saf., 21, 250-257 (2006).
7 Hong, C.K., Seo, Y.H., Choi, C.M., Hwang, I.S., Kim, M.S., Microbial quality of fresh vegetables and fruit in Seoul, Korea. J. Food Hyg. Saf., 27, 24-29 (2012).   DOI
8 Augustynowicz, E., Gzyl, A., Slusarczyk, J., Detection of enterotoxigenic Clostridium perfringens with a duplex PCR. J. Med. Microbiol., 51, 169-172 (2002).   DOI
9 Park, S.H., Choi, O.K., Jeong, J.A., Kim, W.H., Lee, Y.E., Park, K.H., Yoon, M.H., Genetic diversity of Clostridium perfringens form food-poisoning outbreak in Gyeonggi-do 2013-2014. Korean J. Microbiol., 52, 286-297 (2016).   DOI
10 Brynestad, S., Granum, P.E., Clostridium perfringens and foodborne infections. Int. J. Food Microbiol., 74, 195-202 (2002).   DOI
11 Scherer, K., Made, D., Ellerbroek, L., Schelengburg, J., Johne, R., Klein, G., Application of a swab sampling method for the detection of norovirus and rotavirus on artificially contaminated food and environmental surfaces. Food Environ. Virol., 1, 42-49 (2009).   DOI
12 Kim, D.K., Kim, S.J., Kang, D.H., Comparison of a four-section spindle and stomacher for efficacy of detaching microorganisms from fresh vegetables. J. Food Prot., 78, 1380-1386 (2015).   DOI
13 Sharpe, A.N., Jackson, A.K., Stomaching a new concept in bacteriological sample preparation. Appl. Microbiol., 24, 175-178 (1972).   DOI
14 Moret, S., Conte, L.S., High-performance liquid chromatographic evaluation of biogenic amines in foods an analysis of different methods of sample preparation in relation to food characteristics. J. Chromatogr. A, 729, 363-369 (1996).   DOI
15 Miki, Y., Miyamoto, K., Ikuko, K.H., Fujiuchi, K., Akimoto, S., Prevalence and characterization of enterotoxin gene-carrying Clostridium perfringens isolates from retail meat products in Japan. Appl. Environ. Microbiol., 74, 5366-5372 (2008).   DOI
16 Kim, S.R., Yoon, Y.H., Kim, W.I., Park, K.H., Yun, H.J., Chung, D.H., Yun, J.C., Ryu, K.Y., Comparison of sample preparation methods for the recovery of foodborne pathogens from fresh produce. J. Food Prot., 75, 1213-1218 (2012).   DOI
17 Wilson, I.G., Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol., 63, 3741-3751 (1997).   DOI
18 Reischl, U., Linde, H.J., Metz, M., Leppmeier, B., Lehn, N., Rapid identification of methicillin-resistant Staphylococcus aureus and simultaneous species confirmation using realtime fluorescence PCR. J. Clin. Microbiol., 38, 2429-2433 (2000).   DOI
19 Sepp, R., Szabo, I., Uda, H., Sakamoto, H., Rapid techniques for DNA extraction from routinely processed archival tissue for use in PCR. J. Clin. Pathol., 47, 318-323 (1994).   DOI
20 De Medici, D., Croci, L., Delibato, E., Di Pasquale, S., Filetici, E., Toti, L., Evaluation of DNA extraction methods for use in combination with SYBR Green I real-time PCR to detect Salmonella enterica serotype Enteritidis in poultry. Appl. Environ. Microbiol., 69, 3456-3461 (2003).   DOI
21 Giacomazzi, S., Leroi, F., Joffraud, J.J., Comparison of three methods of DNA extraction from cold-smoked salmon and impact of physical treatments. J. Appl. Microbiol., 98, 1230- 1238 (2005).   DOI
22 Quigley, L., O'Sullivan, O., Beresford, T.P., Paul Ross, R., Fitzgerald, G.F., Cotter, P.D., A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese. J. Appl. Microbiol., 113, 1364-5072 (2012).