• International Journal of Oral Biology
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• v.46 no.3
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• pp.140-145
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• 2021

This study aimed to develop Lautropia mirabilis-specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis. The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

• Applied Biological Chemistry
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• v.49 no.3
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• pp.192-195
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• 2006

For the development of a qualitative PCR detection method for genetically modified perilla (Perilla frutescens), perilla species-specific gene, KAS-I (Beta-ketoacyl-ACP synthase I), was selected and validated as suitable for the use as an endogenous reference gene in perilla. Primer specificity was first tested by the means of qualitative PCR analysis. The primer pair Pfru3-F/R amplifying the perilla endogenous gene, KAS-I, gave rise to an amplicon 95 bp. No amplified product was observed when DNA samples from 15 different plants were used as templates. Qualitative PCR detection method was assayed with vitamin E-enriched GM Perilla developed in Korea. For the qualitative PCR detection method, the construct-specific detection primer pairs were constructed. The primer pair TMTO-F/R amplifying the junction region of TMT (${\gamma}$-tocopherol methyltransferase) gene and OCS (Octopine synthase) terminator introduced in GM perilla gave rise to an amplicon 148 bp.

• Food Science of Animal Resources
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• v.29 no.5
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• pp.647-652
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• 2009

A duplex PCR technique was applied for specific identification of cow and goat milk in milk products by using primers targeting the mitochondrial 12S rRNA gene. Duplex PCR using primers specific for cow and goat generated specific fragments of 223bp and 326bp from cow and goat milk DNA, respectively. Duplex PCR was applied to 15 milk products purchased from the market to verify label statements. The labeling statements of four market milk products, three yoghurt products, and one whole milk powder product were confirmed in the duplex PCR. The labeling statements of five of seven infant milk powder products were also confirmed by duplex PCR but the other two products were shown to be contaminated with either cow or goat milk. The proposed duplex PCR provides a rapid and sensitive approach to detection of as little as 0.1% cow milk in goat milk and one-step detection of cow or goat milk in milk products.

• Korean Journal of Food Science and Technology
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• v.31 no.6
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• pp.1628-1634
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• 1999

The polymerase chain reaction (PCR) for the sensitive and specific detection of Listeria monocytogenes was employed by using LM 1 and LM 2 primers which were based on the listeriolysin O gene. The direct use of cell suspension as DNA template, without DNA extraction or lysis step, was suitable and specific enough to detect L. monocytogenes at the level of $10^2$ CFU or less per PCR for the pure culture and milk sample, however, the detection sensitivity became blunt for other food samples such as kimchi and chicken. The nested PCR, in which L-1 and L-2 (both designed from listeriolysin O gene) were employed as inner primers, was specific for detecting L. monocytogenes and enhanced the detection limit by 10 times. The PCR using LM 1 and LM 2 primers was very effective to detect L. monocytogenes from foods in terms of the specificity and time consumed, i. e. within $4{\sim}8\;hrs$ (nested PCR).

• Biomedical Science Letters
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• v.9 no.3
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• pp.139-144
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• 2003

Since water borne infection causes acute diseases and results in spread of diseases by secondary infection, the prevention is very important. Therefore, it is necessary to have a method that is rapid and effective to monitor pathogenic bacteria in drinking water. In this study, we employed a systematic method, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, to develop an effective monitoring system for possible bacterial contaminants in drinking water. For this purpose, PCR primers were derived from 992 bp region of the 16s rRNA gene that is highly conserved through the different species of prokaryotes. To test whether the PCR primers designed are indeed useful for detecting all the possible microbial contaminants in the water, the primers were used to amplify 16s rRNA regions of different microbial water-borne pathogens such as E. coli, Salmonella, Yersinia, Listeria, and Staphylococcus. As expected, all of tested microorganisms amplified expected size of PCR products indicating designed PCR primers for 16s rRNA indeed can be useful to amplify all different microbial water-borne pathogens in the water. Furthermore, to test whether these 16s rRNA based PCR primers can detect bacterial populations present in the water, water samples taken from diverse sources, such as river, tap, and sewage, were used for amplification. PCR products were for then subjected for cloning into a T-vector to generate a library containing 16s rRNA sequences from various bacteria. With cloned PCR products, RFLP analysis was done using PCR products digested with restriction enzyme such as Hae III to obtain species-specific RFLP profiles. After PCR-RFLP, the bacterial clones which showed the same RFLP profiles were regarded as the same ones, and the clones which showed distinctive RFLP profiles were subsequently subjected for sequence analysis for species identification. By this PCR-RFLP analysis, we were able to reveal diverse populations of bacteria living in water. In brief, in unsterilized natural river water, over 60 different species of bacteria were found. On the other hand, no PCR products were detected in drinking tap-water. The results from this study clearly indicate that the PCR-RFLP-sequence analysis can be a useful method for monitoring diverse, perhaps pathogenic bacteria contaminated in water in a rapid fashion.

• Proceedings of the Korean Vacuum Society Conference
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• 2016.02a
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• pp.375.1-375.1
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• 2016

Polymerase chain reaction (PCR) has revolutionized genetics and become one of the most popular techniques in modern biological and medical sciences. It can be used not only as an in vitro DNA amplification method but also used in many bioassay applications. The PCR can be used to exponentially produce a large number of DNA copies from a small quantity of DNA molecules in a few hours. However, as unwanted DNA fragments are also often manufactured, the amplification efficiency of PCR is decreased. To overcome this limitation, several nanomaterials have been employed to increase the specificity of the PCR reaction. Recently, graphene has attracted a great interest for its excellent electron transfer, thermal and biocompatibility. Especially, gold nanoparticle-coated graphene oxide (GO/AuNPs) led to enhance electron and thermal transfer rate and low-charge transfer resistance. Therefore, we report the development of a demonstration for the PCR efficiency using a large-scale production of the GO and combination of gold nanoparticles. Because a thermal conductivity is an important factor for improving the PCR efficiency in different DNA polymerases and different size samples. When PCR use GO/AuNPs, the result of transmission electron microscopy and real-time quantitative PCR (qPCR) showed an enhanced PCR efficiency. We have demonstrated that GO/AuNPs would be simply outperformed for enhancing the specificity and efficiency of DNA amplification procedure.

• BMB Reports
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• v.37 no.3
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• pp.356-361
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• 2004

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.

• International Journal of Oral Biology
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• v.40 no.4
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• pp.205-210
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• 2015

Prevotella intermedia-specific quantitative real-time PCR (qPCR) primers were previously designed based on the nucleotide sequences of RNA polymerase ${\beta}$-subunit gene (rpoB). However, the several clinical strains isolated from Korean populations are not detectable by the qPCR primers. The purpose of this study was to develop new P. intermedia-specific qPCR primers based on the rpoB. The specificity of the primers was determined by conventional PCR with 12 strains of P. intermedia and 52 strains (52 species) of non-P. intermedia bacteria. The sensitivity of primers was determined by qPCR with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of P. intermedia ATCC $25611^T$. The data indicated that only P. intermedia strains were detected by the P intermedia-specific qPCR primers (RTPiF2/RTPiR2); in addition, as little as 40 fg of P. intermedia genomic DNA could be detected. These results suggest that these qPCR primers are useful in detecting P. intermedia from the bacterial infectious lesions including dental plaque and oral tissue lesions.

• Food Science of Animal Resources
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• v.30 no.4
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• pp.590-596
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• 2010

Meat and meat products are a potential source of food-borne pathogens, including Staphylococcus aureus, Salmonella spp., Escherichia coli O157:H7, and Bacillus cereus. A sensitive and specific PCR assay for the detection of these pathogens in meat and meat products was developed in this study, as part of a broader effort to reduce the potential health hazards posed by these pathogens. Initially, PCR conditions were standardized with purified DNA. Under standard conditions, the detection level for PCR was as low as 10 pg of purified bacterial DNA. After overnight growth of bacteria in a broth medium, as few as $10^2$ CFU of bacteria were detected by PCR assay. The primers employed in the PCR assay were found to be highly specific for individual organisms, and evidenced no cross-reactivity with heterologous organisms. Additionally, the multiplex PCR assays also amplified some target genes from the four pathogens, and multiplex amplification was obtained from as little as 10 pg of DNA, thus illustrating the excellent specificity and high sensitivity of the assay. In conclusion, this PCR-based technique provides a sensitive and specific method for the detection of S. aureus, Salmonella spp., E. coli O157:H7, and B. cereus in meat and meat products.

• Korean Journal of Microbiology
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• v.41 no.3
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• pp.168-176
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• 2005

Vibrio vulnificus, one of the marine bacterial pathogens causing septicemia, was detected using molecular methods, namely, PCR and/or Southern hybridization, and real-time PCR. Extracted and purified total DNAs by using commercial kits were used as templates for PCR. Multiplex-PCR was conducted by employing three sets of primers for the genes, hemolysin (vvhA), phosphomannomutase (pmm), and metalloprotease (vvpE), for V vulnificus virulence. The presence of DMSO ($5\%$) and BSA ($0.1\%$) in PCR reaction mixture improved a detection efficiency by higher PCR band intensities. TaqMan real-time PCR was carried out by using gene segment of vvhA as a target. Detection limit of PCR/Southern hybridization without enrichments was to be around $10^2\;cells\;g^{-1}$ of sample. However, those three methods using the enrichment at $35^{\circ}C$ in APW showed high sensitivity ($2\~10\;cells\;g^{-1}$ of sediments). Highly sensitive detection of V vulnificus by real-time PCR was achieved within $5\~6$ hr, whereas the detection by PCR/Southern hybridization required about 36 hr. Thus, it was evident that real-time PCR is the most rapid and efficient method for detecting V vulnificus in tidal flat sediments.