• Korean Journal of Fisheries and Aquatic Sciences
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• v.35 no.5
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• pp.490-496
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• 2002

In order to detect the DNA heteropolymorphism of chum salmon, selected essential genes were examined in different regional chum salmons, i.e., Korean, Japanese and American by denaturing gradient gel electrophoresis (DGGE) and real time PCR methods. From the promoter regions and introns of growth hormone, mtDNA NDI region, D-loop region, IGF-I, histone H3 and MCH2 several representative primer pairs were obtained and employed for the DGGE with the PCR products from the genomic DNAs of the different regional chum salmons. mtDNA NDI, D-loop region and IGE-I genes showed marked heteropolymorphism between Korean and American chum salmons. Intron C of growth hormone also showed a heteropolymorphism between Korean and Japanese chum salmons. Whereas heteropolnnorphism of histone liH and MCH2 genes was detected among in Korean, Japanese and Asnerican chum salmons in the examined region. The real time PCR disclosed the characteristic incremental production of target DNAs dependent on the heteropolymorphic conditions of genomic DNAa of chum salmons, thus the different regional chum salmons could be grouped by the variable incremental curies. Although the DGGE and real time PCR did not produce the identical results in this study, we suggest that the DGGE and real time PCR could be used for the primary screening of the DNA heteropolymorphism of different animal genome.

• Korean Journal of Veterinary Research
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• v.44 no.4
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• pp.655-662
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• 2004

The aim of this work was the validation of a rapid real-time PCR assay based on TaqMan technology for the unequivocal identification of infectious canine hepatitis (ICH) virus, to be used directly on DNA purified from blood specimens. A real-time PCR system targeting at the E3 ORFA gene sequence of canine adenovirus type 1 was optimized and validated through comparative analysis of samples using conventional PCR system. The real-time PCR assay based on TaqMan technology could disclose 23 (37.7%) out of 61 samples as PCR positive. In contrast, 18 (29.5%) samples were found PCR positive when conventional PCR was applied on these samples. The use of the ABI Prism 7700 sequence detection system allowed the efficient determination of the amplified product accumulation through a fluorogenic probe. The entire real-time TaqMan PCR assay, including DNA extraction, amplification, and detection could be completed within 3 hours. The detection method of real-time TaqMan PCR assay was 1,000 times more sensitive than conventional PCR. Real-time TaqMan probe and primer set developed and optimized in this study is a sensitive, rapid and accurate method for detection of ICH virus and can be effective screening tool for the detection of ICH in a diagnostic laboratory routines.

• Korean Journal of Food Science and Technology
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• v.42 no.3
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• pp.355-360
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• 2010

Vibrio parahaemolyticus (V. parahaemolyticus), which is commonly found in raw seafood, causes gastroenteritis in humans. Rapid and effective methods have been developed as culture methods require up to 5-7 days. In this study, real-time PCR was compared with the standard culture method for detecting V. parahaemolyticus in seafood and radish sprout samples. Five hundred grams of the samples were artificially contaminated with V. parahaemolyticus then divided into 20 samples. The samples were incubated in alkaline peptone water and then streaked onto thiosulfate-citrate-bile saltssucrose agar. Biochemical tests for suspicious colonies were performed using the API 20NE strip. In parallel, real-time PCR was performed targeting the toxR gene using the enrichment broth. The real-time PCR was sensitive in discriminating V. parahaemolyticus from other foodborne pathogens. The detection limit of the real-time PCR was $10^3\;CFU/mL$ in phosphate-buffered saline. Although the real-time PCR detected more positive samples (76 out of 180, 42%) than the culture method (66 out of 180, 37%), there was no significant statistical difference (p>0.05) between the two methods. In conclusion, real-time PCR assays could be an alternative to the standard culture method for detecting V. parahaemolyticus in seafood and radish sprouts, which has many advantages in terms of detection time, labor, and sensitivity.

• Korean Journal of Veterinary Service
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• v.34 no.4
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• pp.321-331
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• 2011

Mycobacterium bovis, a member of the M. tuberculosis complex (MTC), is the causative agent of bovine tuberculosis. Detection of M. bovis and M. tuberculosis using conventional culture- and biochemical-based assays is time-consuming. Therefore, a simple and sensitive molecular assay for rapid detection would be of great help in specific situations such as faster diagnosis of bovine tuberculosis (bTB) infection in the abattoirs. We developed a novel multiplex real-time PCR assay which was applied directly to biological samples with evidence of bTB and it was allowed to differentiate between M. bovis and M. tuberculosis. The primers and TaqMan probes were designed to target the IS1081 gene, the multi-copy insertion element in the MTC and the 12.7-kb fragment which presents in M. tuberculosis, not in the M. bovis genome. The assay was optimized and validated by testing 10 species of mycobacteria including M. bovis and M. tuberculosis, and 10 other bacterial species such as Escherichia coli, and cattle lymph nodes (n=113). The tests identified 96.4% (27/28) as M. bovis from the MTC-positive bTB samples using conventional PCR for specific insertion elements IS1081. And MTC-negative bTB samples (n=85) were tested using conventional PCR and the real-time PCR. When comparative analyses were conducted on all bovine samples, using conventional PCR as the gold standard, the relative accuracy of real-time PCR was 99.1%, the relative specificity was 100%, and the agreement quotient (kappa) was 0.976. The detection limits of the real-time PCR assays for M. bovis and M. tuberculosis genomic DNA were 10 fg and 0.1 pg per PCR reaction, respectively. Consequently, this multiplex real-time PCR assay is a useful diagnotic tool for the identification of MTC and differentiation of M. bovis and M. tuberculosis, as well as the epidemiologic surveillance of animals slaughtered in abattoir.

• Korean Journal of Breeding Science
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• v.43 no.5
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• pp.448-456
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• 2011

We used suppression subtractive hybridization (SSH) combined with mirror orientation selection (MOS) method to screen differentially expressed genes from red-fleshed kiwifruit 'Hongyang'. As a result, the 288 clones were obtained by subcloning PCR product and 192 clones that showed positive clones on colony PCR analysis were selected. All the positive clones were sequenced. After comparisons with the NCBI/Genbank database using the BLAST search revealed that 30 clones showed sequence similarity to genes from other organisms; 10 clones showed significant sequence similarity to known genes. Among these clones, 3 clones (AcF21, AcF42 and AcF106) had sequence homology to 1-aminicyclopropane-carboxylic acid (ACC)-oxidase (ACO) that known to be related to fruit ripening. The expression patterns of differentially expressed genes were further investigated to validate the SSH data by reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qReal-time PCR) analysis. All the data from qReal-time PCR analysis coincide with the results obtained from RT-PCR analysis. Three clones were expressed at higher levels in 'Hongyang' than 'Hayward'. AcF21 was highly expressed in the other genes at 120 days after full bloom (DAFB) and 160 DAFB of 'Hongyang'.

• Food Science of Animal Resources
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• v.29 no.4
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• pp.506-512
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• 2009

Salmonellosis is an important worldwide foodborne infectious disease that is transmitted by many food vehicles including raw and processed animal products and fresh produce. In this study, the effectiveness of automated ELISA ($VIDAS^{(R)}$) and realtime PCR in the detection of Salmonella spp. in steamed pork and raw broccoli sprouts was evaluated by comparing their results with those of a conventional culture method. Bulk samples (500 g) of steamed pork and raw broccoli sprouts were inoculated with various levels of Salmonella and divided into 20 samples (25 g each). All the samples, including the controls, were analyzed using a conventional culture method, $VIDAS^{(R)}$, and real-time PCR to detect the presence of Salmonella. In addition, the levels of background flora in the steamed pork and the raw broccoli sprouts were determined. In the steamed pork that contained less than 100 CFU/g of aerobic bacteria, all three methods detected low levels of Salmonella without a statistical difference in their performance. In the broccoli sprouts with high quantities of background flora (ca. $6.7{\times}10^7$ CFU/g), however, all three methods were unable to detect low levels of Salmonella, and real-time PCR and $VIDAS^{(R)}$ more sensitively detected Salmonella than the culture method, with significant statistical differences. In conclusion, $VIDAS^{(R)}$ and real-time PCR could be superior to conventional culture methods in detecting Salmonella in food with high levels of background flora.

• Korean Journal of Environmental Agriculture
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• v.30 no.4
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• pp.367-376
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• 2011

BACKGROUND: In order to develop effective assessment method for Korean paddy soil microbial community structure, reliable genomic DNA extraction method from paddy soil and quantitative real-time PCR (qRT-PCR) method are needed to establish METHODS AND RESULTS: Out of six conventional soil genomic DNA extraction methods, anion exchange resin purification method was turn to be the most reliable. Various PCR primers for distinguishing five bacterial phylum (${\alpha}$-Proteobacteria, ${\beta}$-Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes), all bacteria, and all fungi were tested. Various qRT-PCR temperature conditions were also tested by repeating experiment. Finally, both genomic DNA extraction and qRT-PCR methods for paddy soil were well established. CONCLUSION: Quantitative real-time PCR (qRT-PCR) method to assess paddy soil microbial community was established.

• Microbiology and Biotechnology Letters
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• v.36 no.1
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• pp.34-42
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• 2008

Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biologics such as biopharmaceuticals, tissue engineered products, and cell therapy. Manufacturing processes for the biologics using bovine materials have the risk of viral contamination. Therefore viral validation is essential in ensuring the safety of the products. Bovine viral diarrhoea virus (BVDV) is the most common bovine pathogen and has widely been known as a contaminant of biologics. In order to establish the validation system for the BVDV safety of biologics, a real-time RT-PCR method was developed for quantitative detection of BVDV contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BVDV RNA was selected, and BVDV RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 1 $TCID_{50}/mL$. The rent-time RT-PCR method was validated to be reproducible and very specific to BVDV. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BVDV. BVDV RNA could be quantified in CHO cell as well as culture supernatant. Also the real-time RT-PCR assay could detect $10TCID_{50}/mL$ of BVDV artificially contaminated in bovine collagen.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.10
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• pp.1522-1527
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• 2010

This study was conducted to find out the minimal time needed for detection of Salmonella spp. which exist at very low concentration in foods by using real-time PCR. The sal-F and sal-R sequences were used as primers and sal-P was used as a probe. The detection limit of Salmonella spp. was $3.77{\times}10^2\;cfu/mL$ in buffered peptone water (BPW). Microbial growth was monitored after artificially inoculated Salmonella spp. into BPW. The obtained growth curve was well fitted with the equation, y＝$0.0127x^2$+0.5927x-0.4317 ($R^2$=0.99), if assuming that 1 cell exists in 25 g sample (0.04 cfu/mL). The microbial concentration will be reduced to 10 fold by adding BPW during sample treatment, so actual initial concentration at the starting point of enrichment is 0.004 cfu/mL. At this condition, real-time PCR detection would be possible only when microbial concentration increase occurs to exceed the detection limit (377 cfu/mL). The time needed for microbial increase was calculated from the growth curve equation as 7 hours and 20 minutes. Therefore the total time required for detection was less than 10 hours including the PCR operating time.

• Food Science and Preservation
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• v.30 no.2
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• pp.262-271
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• 2023

The objective of this study is to compare and assess the effectiveness of real-time polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), and the selective agar plate method for the detection of Salmonella spp. and Listeria monocytogenes in ready-to-eat (RTE) foods. In RTE foods, the detection performance of the three methods (RT-PCR [SureTect^TM kit and PowerChek^TM kit], LAMP [3M MDS], selective agar) were similar at 0-10, 10-50, 50-100, and 100- CFU/mL of Salmonella spp. and L. monocytogenes. We found that with RT-PCR, the Ct value of salad was significantly higher (p<0.05) than other RTE foods, indicating that fiber plays a critical role as an obstacle to the rapid detection of Salmonella spp. However, the Ct value displayed a mixed pattern according to the inoculation level of L. monocytogenes. The use of rapid detection kits and machines mostly depends on the user's choice, with accuracy, ease of use, and economy being the primary considerations. As an RT-PCR kit, SureTect^TM and PowerChek^TM showed high accuracy in detecting Salmonella spp. and L. monocytogenes in RTE foods, showing that they can replace the existing RT-PCR kits available. Additionally, LAMP also showed excellent detection performance, suggesting that it has the potential to be used as a food safety management tool.