Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay |
| |
| Authors: | Ji-Yeon Hyeon In-Gyun Hwang Hyo-Sun Kwak Chankyu Park In-Soo Choi Kun-Ho Seo |
| |
| Institution: | 1Department of Public Health, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Korea.;2Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Korea.;3Korea Food and Drug Administration, Seoul 122-704, Korea.;4Department of Animal Biotechnology, College of Animal Bioscience and Technology, Konkuk University, Seoul 143-701, Korea. |
| |
| Abstract: | The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method. |
| |
| Keywords: | enrichment broth inhibition real-time PCR Salmonella |
|
|
