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

• Journal of Life Science
• /
• v.27 no.7
• /
• pp.746-753
• /
• 2017

In order to rapidly identify four drums species, Larimichthys polyactis, L. crocea, Atrobucca nibe, and Pseudotolithus elongates, a highly efficient and quick method has been developed using multiplex polymerase chain reaction (PCR) with species-specific primers. Around 1.4 kbp of the mitochondrial COI gene sequences from the four drums species were aligned, and species-specific forward primers were designed, based on the single nucleotide polymorphism (SNP). The optimal conditions for PCR amplification were selected through cross-reactivity, using a gradient PCR method. The PCR results demonstrated species-specific amplification for each species at annealing temperatures between 50 and $62^{\circ}C$. Multiplex species-specific PCR (MSS-PCR) amplification reactions with four pairs of primers were performed for sixteen specimens of each species. MSS-PCR lead to a species-specific amplification of a 1,540 bp fragment in L. polyactis, 1,013 bp in A. nibe, 474 bp in L. crocea, and 182 bp in P. elongates, respectively. The four different sizes of each PCR product can be quickly and easily detected by single gel electrophoresis. The sensitivity of the MSS-PCR of the DNA was up to $0.1ng/{\mu}l$ as a starting concentration for the four different species tested. These results suggest that MSS-PCR, with species-specific primers based on SNP, can be a powerful tool in the rapid identification of the four drums species, L. polyactis, L. crocea, A. nibe, and P. elongates.

• Korean Journal of Microbiology
• /
• v.41 no.1
• /
• pp.60-66
• /
• 2005

To select the rapid and efficient molecular subtyping method for epidemiologic monitoring of Staphylococcus aureus (S. aureus) strains at clinical laboratory levels, a total 116 of S. aureus and MRSA (methicillin-resistant S. aureus) strains from diverse animal species [Korean cattle, goat, pig, dog, chicken, mouse] and also humans were analyzed. To evaluate the discriminatory ability (DA) of individual PCR methods, random amplified polymorphic of DNA [RAPD; 4M & RA primer], repetitive extragenic palindromic sequences PCR (REP-PCR), and enterobacterial repetitive intergenic consensus sequences PCR (ERIC-PCR) methods were conducted and then compared on their Simpson's index of diversity (SID) values based on the dendrogram patterns, which was produced by software program (BiolD2+ & GelCompar II). In first, RAPD using the 4M primer (SID 0.915) was expressed more higher SID value than that of RA primer (SID 0.874). 4M primer was expressed more powerful DA than RA. Both REP-PCR (SID 0.930) and ERIC-PCR (SID 0.929) methods showed much more higher DA than that of RAPD. According to the present results, both REP-PCR and ERIC-PCR among the tested analysis methods were found as the most reliable and discriminative molecular subtyping method, because they expressed the highest DA for the present S. aureus and MRSA strains.

• Applied Biological Chemistry
• /
• v.50 no.4
• /
• pp.276-280
• /
• 2007

Buckwheat often causes severe allergic reactions in sensitive people. One of the major allergenic proteins in common buckwheat (Fagopyrum esculentum) has been found to be a BW10KD protein. In this study, we developed a PCR method to detect buckwheat ingredients in food using primers corresponding to the allergenic BW10KD gene. Five pairs of oligonucleotide primers successfully enabled PCR amplification of the specific regions of the genomic BW10KD DNA from buckwheat, but no amplification from seven other cereals and beans (barley, wheat, German millet, African millet, soybean, red bean, and black bean). The proposed PCR method was applied to analyze 12 processed foods (buckwheat flour, buckwheat noodle, buckwheat jelly, wheat noodle, instant noodle, black sesame gruels, sunsik, cookie, misutkaru, and three kinds of cereal); among them, only three samples including buckwheat flour, buckwheat noodle and buckwheat jelly showed a positive reaction to the detection. This PCR method was able to detect as little as 1 ng of common buckwheat DNA. This rapid and specific PCR method would be applicable to detect allergenic buckwheat ingredients in food.

• Food Science of Animal Resources
• /
• v.27 no.2
• /
• pp.209-215
• /
• 2007

In order to develop a sensitive and reliable method for the species-specific molecular markers, PCR-RFLP assay of the mitochondrial DNA(mt DNA) 12S rRNA gene was exploited for the identification of the origin of animal meat species including cattle, pig, sheep, goat, horse, deer, chicken, duck and turkey. A specific primer pairs were designed, based on the nucleotide sequences of mt 12S rRNA gene, for the amplification of the highly conserved region in the gene of the animal species using PCR-RFLP technique. mt DNA was isolated from meat samples followed by DNA amplification using PCR with the specific primers. PCR amplification produced an approximately 455 bp fragment in each of these animal meats. To distinguish pleat species, the PCR amplicons were digested with restriction endonucleases Tsp5091 and MboI, respectively, which generates distinct RFLP profiles. The DNA profiles digested with Tsp5091 allowed the clear discrimination in the mammalian meat species and the DNA profiles digested with MboI in poultry meat species. Therefore, the PCR-RFLP profiles of mt 12S rRNA gene could be very useful to identify the origin of the raw materials in the raw meats as well as the processed meat products.

• Parasites, Hosts and Diseases
• /
• v.55 no.6
• /
• pp.601-606
• /
• 2017

Cystoisospora is responsible for morbidity in immunocompromised patients. PCR is sensitive for diagnosing Cystoisospora; however, it needs reevaluation for differential molecular diagnosis of cystoisosporiasis. We aimed at evaluating melting curve analysis (MCA) after real-time PCR (qPCR) in diagnosis and genotyping of Cystoisospora as an alternative to conventional PCR. We included 293 diarrheic stool samples of patients attending the Department of Clinical Oncology and Nuclear Medicine of Cairo University Hospitals, Egypt. Samples were subjected to microscopy, nested PCR (nPCR), and qPCR targeting the internal transcribed spacer 2 region (ITS2) of the ribosomal RNA (r RNA) gene followed by melting temperatures ($T_ms$) analysis and comparing the results to PCR-RFLP banding patterns. Using microscopy and ITS2-nPCR, 3.1% and 5.8% of cases were Cystoisospora positive, respectively, while 10.9% were positive using qPCR. Genotyping of Cystoisospora by qPCR-MCA revealed 2 genotypes. These genotypes matched with 2 distinct melting peaks with specified $T_ms$ at $85.8^{\circ}C$ and $88.6^{\circ}C$, which indicated genetic variation among Cystoisospora isolates in Egypt. Genotype II proved to be more prevalent (65.6%). HIV-related Kaposi sarcoma and leukemic patients harbored both genotypes with a tendency to genotype II. Genotype I was more prevalent in lymphomas and mammary gland tumors while colorectal and hepatocellular tumors harbored genotype II suggesting that this genotype might be responsible for the development of cystoisosporiasis in immunocompromised patients. Direct reliable identification and differentiation of Cystoisospora species could be established using $qPCR-T_ms$ analysis which is useful for rapid detection and screening of Cystoisospora genotypes principally in high risk groups.

• Korean Journal of Veterinary Research
• /
• v.47 no.4
• /
• pp.399-407
• /
• 2007

Cause of high lethality and dissemination to human being, new development of rapid method for the detection of highly pathogenic Avian Influenza Virus (AIV) is still necessary. For the detection of AIV subtype H5N1, typical pathogenic AIV, new method to confirm sub-typing of this virus is also needed. For the purpose of ultra-rapid detection and sub-typing of hemagglutinin and neuraminidase of AIV, this study was planned. As the results we could demonstrate an ultra-rapid multiplex real-time PCR (URMRT PCR) for the detection of AIV In this study, the URMRT PCR were optimized with synthesized AIV H5- and AIV Nl-specific DNA templates and GenSpector TMC, which is a semiconductor process technology based real-time PCR system with high frequencies of temperature monitoring. Under eight minutes, the amplifications of two AIV subtype-specific PCR products were successfully and independently detected by 30 cycled ultra-rapid PCR, including melting point analysis, from $1{\times}10^3$ copies of mixed template DNA. The URMRT PCR for the detection of AIV H5N 1 developed in this study could be expected to apply not only detections of different AIVs, but also various pathogens. It was also discussed that this kind of the fastest PCR based detection method could be improved by advance of related technology in near future.

• Journal of Animal Science and Technology
• /
• v.46 no.5
• /
• pp.841-848
• /
• 2004

Rapid detection of viable Salmonella in pasteurized milk is important to protect public health from food poisoning. Reverse transcriptase-polymerase chain reaction(RT-PCR) is recognized as a molecular genetical method to differentiate between live and dead bacteria The RT-PCR in this study was designed to detect specifically viable Salmonella in milk by using the primers whose nucleotide sequences were determined based on fimA gene which encodes the submit of type 1 fimbriae. Treatment of RNA preparation with RNase-free DNase was adequate enough to destroy DNA, which may otherwise be amplified in the RT PCR Seven strains of Salmonella were detected in the RT-PCR but Escherichia coli, Shigella sonnei, Citrobacter freundii, and Klebsiella pneumoniae were not. $10^7/ml$ and $10^6/ml$ of dead Salmonella which were heat-treated in milk were detectable by using the RT-PCR but $10^5{\sim}10/ml$ of the dead bacteria were not. The sensitivity of the RT-PCR in detecting viable Salmonella was 100 cells/ml.

• Korean Journal of Plant Resources
• /
• v.14 no.3
• /
• pp.235-240
• /
• 2001

Polymerase chain reaction (PCR) is used in a wide array of researches in plant molecular genetics and breeding. However, considerable time and cost are still required for the preparation of DNA suitable for reliable PCR results, especially in plant species containing high amounts of polyphenols. To reduce time and effort for PCR-based analysis, a simplified but reliable method was developed by a combinational employment of a simple and fast DNA extraction procedure and BLOTTO (Bovine Lacto Transfer Technique Optimizer) in reaction mixture. Genomic DNAs prepared by one-step extraction method from recalcitrant plant species such as Rubus coreanus, apple, grape and lettuce were successfully amplified by random primers in the reaction mixture containing 2 to 4% BLOTTO. Successful amplification of ${\gamma}$-TMT transgene in lettuce transformants by the specific primers was also achieved in the same condition, making rapid screening of positive transformants possible. Our results suggest that use of a simple DNA extraction procedure and incorporation of BLOTTO in reaction mixture in combination can reduce time and effort required for the analyses of a large number of germplasms and transformants by PCR-based techniques.

• Korean Journal of Agricultural Science
• /
• v.38 no.1
• /
• pp.45-50
• /
• 2011

This study was conducted to investigate the farm situation about bovine leukemia virus(BLV) infection that greatly influence productivity in dairy cattle and compare the accuracy of diagnosis for BLV infection between PCR and ELISA techniques. Blood samples of 193 heads from 5 herds in Chungnam and Chungbuk area were used to analyze BLV gene and serum, and the results were obtained as follows. The amplified BLV gene in dairy cattle by PCR technique resulted in 226 bp, 596 bp and 434 bp, respectively, for gag, pol and env, which were well amplified. The infection rates of BLV virus diagnosed by PCR and ELISA techniques ranged from 80.55 to 100% and from 22.22 to 86.95%, respectively, and the infection rates among 5 herds were significantly different in both methods (P<0.05). Further, the average infection rates of 5 herds were 87.05 and 63.21%, respectively, for PCR and ELISA techniques. Kappa statistics for examining consistency of diagnosis by PCR and ELISA techniques showed 0.246, which represents low consistency. Consequently, PCR based BLV technique was considered as a corrective measure for diagnosis of BLV infection in Holstein dairy cattle.