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

A loop‐mediated isothermal amplification (LAMP) method for rapid detection of enterotoxigenic Clostridium perfringens in food is developed and evaluated in this study. Six primers were designed to recognize the cpa gene of C. perfringens. A panel of 45 bacterial strains, including 15 C. perfringens and 30 other strains, were included in this study to evaluate and optimize the LAMP assay. The specificity of the LAMP assay was 100%. The sensitivity of the LAMP assay for the detection of C. perfringens in food was 10 CFU/ml. Different methods of extracting DNA from artificially contaminated food, such as boiling, NaOH treatment, and the use of magnetic beads, were evaluated. The established LAMP assay was used to analyze C. perfringens in various food samples. The magnetic bead‐based method was a useful tool for extracting genomic DNA from 14 foods contaminated by C. perfringens. All 15 foods contaminated by C. perfringens were identified as positive. Practical Applications The traditional method for detecting Clostridium perfringens in foods involves the use of a bioassay that is laborious and time‐consuming. The LAMP assay is a useful and powerful tool for the rapid detection of C. perfringens, and, undoubtedly, the efficiency, technical simplicity, and cost‐effectiveness of the LAMP assay will have broad applications in bacteriological detection of enterotoxigenic C. perfringens.

Section: Methodsmentioning

confidence: 99%

Development and application of the loop‐mediated isothermal amplification assay for rapid detection of enterotoxigenic Clostridium perfringens in food

Hong¹

2017

Journal of Food Safety

“…All samples were purchased from a local retail market in Seoul, Korea. A mesophilic aerobic plate count was performed for uninoculated food samples to enumerate the background microflora in experimental food samples according to a previously described method ( Chon et al ., 2010 ).…”

Section: Methodsmentioning

confidence: 99%

Comparison of Culture, Conventional and Real-time PCR Methods for Listeria monocytogenes in Foods

Chon

Korean Journal for Food Science of Animal Resources

et al. 2014

Self Cite

We compared standard culture methods as well as conventional PCR and real-time PCR for the detection of Listeria monocytogenes (L. monocytogenes) in milk, cheese, fresh-cut vegetables, and raw beef that have different levels of background microflora. No statistical differences were observed in sensitivity between the two selective media in all foods. In total, real-time PCR assay exhibited statistically excellent detection sensitivity (p<0.05) and was less time consuming and laborious as compared with standard culture methods. Conventional culture methods showed poor performance in detecting L. monocytogenes in food with high levels of background microflora, generating numerous false negative results. While the detection of L. monocytogenes in fresh cut vegetable by culture methods was hindered only by L. innocua, various background microflora, such as L. innocua, L. welshimeri, L. grayi, and Enterococcus faecalis appeared on the two selective media as presumptive positive colonies in raw beef indicating the necessity of improvement of current selective media. It appears that real-time PCR is an effective and sensitive presumptive screening tool for L. monocytogenes in various types of foods, especially foods samples with high levels of background microflora, thus complementing standard culture methodologies.

“…The primer-probe set was designed as described in our previous study (8,22) with the 16S rDNA gene as target. The sequence of the gene was provided by GenBank (accession NR_117066.1).…”

Section: A Ter Ia Ls and M E Th O D Smentioning

confidence: 99%

Rapid Detection of Lactobacillus kefiranofaciens in Kefir Grain and Kefir Milk Using Newly Developed Real-Time PCR

Chon

Journal of Food Protection

et al. 2015

Self Cite

Lactobacillus kefiranofaciens is an indicator microorganism for kefir and a key factor in kefir grain formation and kefiran production. We designed a novel real-time PCR primer and probe set, LKF_KU504, for the rapid detection of L. kefiranofaciens. In inclusivity and exclusivity tests, only 14 L. kefiranofaciens strains were positive among 61 microorganisms, indicating 100 % sensitivity and specificity. The LKF_KU504 set also differentiated kefir milk from 30 commercial nonkefir yogurts. The levels of L. kefiranofaciens in kefir grain and kefir milk were significantly different, indicating L. kefiranofaciens was more concentrated in kefir grain than in kefir milk.