• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.177-181
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• 2003

Eleven bacterial strains which are able to utilize terephthalic acid as a carbon and an energy source for growth were isolated from the soil of 7 water quality evaluation points in Kyonggi area of Korea. Phthalic acid isomer degrading activity of the isolates from the 4 contaminated points was higher than those from the 3 clean points. Among 11 isolates, 4 isolates which have high terephthalic acid degrading activity and degrade two phthalic acid isomers were identified by partal 16S rDNA sequence determination. One of them was identified as Pseudomonas putida, and the others as Comamonas testosteroni. Thus a large number of phthalic acid isomer degrading bacteria in domestic soil were inferred as C. testosteroni. On the basis of these results, the PCR detection of C. testosteroni in soil was applied to monitor soil contamination by phthalic acid isomers. The DNA of C. test-osteroni extracted from 4 g soil was directly detected by PCR with C. testosteroni specific primer pair. The amount of PCR products was different according to sampling sites and more PCR products were obtained from contaminated sites than those from clean sites (Gulpo-chun＞Anyang-chun＞Hwangguji-chun>Shin-chun＞Huk-chun＞Pukhan-river>Kapyeong-chun). This result was coincided with that of the viable cell counts for terephthalic acid degrading bacteria.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.20 no.12
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• pp.117-124
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• 2019

Vibrio cholerae is a very important pathogenic bacterium that has to be monitored in seafood and ships' ballast water. Various methods have been developed to identify this bacterium, yet these methods are time-consuming and have limitations for their sensitivity to detect contamination. The purpose of the present study was to develop a robust and reliable method for identifying V. cholerae. Peptide nucleic acid (PNA) probes were developed to use for PNA-based asymmetrical real-time PCR techniques. The toxigenic Cholera enterotoxin subunit B (ctxB) gene was selected as a target for detecting V. cholerae and the gene was synthesized as a positive template for conventional and real-time PCR. Real-time PCR primers and PNA probes were designed and standard curves were produced for the quantitative analysis. The selected PNA probes reacted specifically to V. cholerae without any ambiguity, even among closely related species, and the detection limit was 0.1 cfu/100 mL. Taken together, the PNA probes and asymmetrical qPCR methods developed in this present study could contribute to the rapid, accurate monitoring of V. cholerae in marine environments, and as well as in seafood and ships' ballast waters.

• Korean Journal of Microbiology
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• v.43 no.2
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• pp.91-99
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• 2007

For the detection of Human Immunodeficiency Virus (HIV), multiple and ultra-rapid real-time PCR methods were developed. The target DNA sequences were deduced from HIV-1 specific 495bp partial env gene (gi_1184090) and from HIV-2 specific 294 bp partial env gene (gi_1332355), and were synthesized by using PCR-based gene synthesis on the reason of safety. Ultra-rapid real-time PCR was performed by $Genspector^{TM}$ using microchip-based, $1\;{\mu}l$ of reaction volume with extremely short time in each 3 step in PCR. The detection including DNA-amplification and melting temperature analysis was completed inner 15 minutes. The HIV-1 specific 117 bp-long and HIV-2 specific 119 bp-long PCR products were successfully amplified from minimum of template,2.3 molecules of each env gene. This kind of real-time PCR was designated as ultra-rapid real-time PCR in this study and it could be applied not only an alternative detection method against HIV, but also other pathogens using PCR-based detection.

• Journal of Korean society of Dental Hygiene
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• v.17 no.2
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• pp.319-330
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• 2017

Objectives: The purpose of this study was to compare SYBR Green qPCR, TaqMan, and bacterial selective medium cultures for accurate quantitative analysis of oral microorganisms. Methods: The SYBR Green method is widely used to analyze the total amount of oral microorganisms in oral saliva. However, in this study, MTR-PCR method based on TaqMan method was performed using newly developed primers and probes. In addition, it was designed to confirm the detection agreement of bacteria among bacteria detection method. Results: As a result of MRT-PCR and SYBR Green qPCR analysis, more than 40 times (0.9-362.9 times) bacterium was detected by MRT-PCR. In addition, more bacteria were detected in saliva in the order of MRT-PCR, SYBR Green qPCR, and bacterium culture, and the results of MRB-PCR and SYBR Green qPCR showed the highest agreement. The agreement between the three methods for detecting P. intermedia was similar between 71.4 and 88.6%, but the agreement between MRT-PCR and SYBR Green qPCR was 80% for S. mutans. Among them, the number of total bacteria, P. intermedia and S. mutans bacteria in saliva was higher than that of SYBR Green qPCR method, and bacterium culture method by MRT-PCR method. P. intermedia and S. mutans in saliva were detected by MRT-PCR and MRT-PCR in 88.6% of cases, followed by the SYBR Green qPCR method (80.0%). Conclusions: The SYBR Green qPCR method is the same molecular biology method, but it can not analyze the germs at the same time. Bacterial culturing takes a lot of time if there is no selective culture medium. Therefore, the MRT-PCR method using newly developed primers and probes is considered to be the best method.

• Journal of Veterinary Clinics
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• v.23 no.3
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• pp.300-307
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• 2006

In the present study, methods of the reverse transcription-polymerase chain reaction(RT-PCR) were evaluated for the rapid detection and differentiation of transmissible gastroenteritis virus(TGEV), porcine epidemic diarrhea virus(PEDV) and rotavirus in piglets suffering from diarrhea. For the purposes, the PCR conditions were first confirmed for the amplification of VP7 gene of rotavirus and N gene of TGEV and PEDV using each specific primers and their annealing temperature. Multiplex RT-PCR methods were further determined to distinguish these viral infections and the results are as follows. For the specific amplification of these viral genes, the reliable PCR condition was determined as 30 cycles of reaction consisting each 1 min of denature at $94^{\circ}C$, annealing at $42^{\circ}C$ and polymerization at $72^{\circ}C$ with 1.0 mM $MgCl_2$. It was able to differentiate these viral infections in the intestines and feces of piglets suffering from diarrhea by duplex PCR for TGEV and PEDV and single PCR for rotavirus with a primer-annealing temperature of $42^{\circ}C$. When the multiplex RT-PCR were undertaken for the field samples, 17 cases of PEDV and 5 cases of rotavirus infections were differential diagnosed in a total of 92 samples of intestines and feces of the piglets with diarrhea.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.30-37
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• 2011

This ultra real-time PCR (UPCR) based diagnosis system for avian influenza A virus (AIV) subtype was designed. The target primer in this study was derived from H5N1 subtype-specific 133 bp partial gene of hemagglutinin (HA), and was synthesized by using PCR-based gene synthesis on the ground of safety. UPCR was operated by Mini-Opticon Q-PCR Quantitative Thermal Cycler using aptamer-based molecular beacon, total 10 ${\mu}l$ of reaction mixture with extraordinarily short time in each steps in PCR. The detection including UPCR and analysis of melting temperature was totally operated within 15 min. The AIV-specific 133 bp PCR product was correctly amplified until 5 molecules of HA gene as minimum of templates. This kind of PCR was drafted as UPCR in this study and it could be used to detect not only AIV subtype, but also other pathogens using UPCR-based diagnosis.

• Journal of Food Hygiene and Safety
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• v.18 no.1
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• pp.6-13
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• 2003

Various kinds of genetically modified organisms (GMO) and processed foods have been developed during recent years. Genetically modified organisms can be classified into several groups as their development methods. Generally, GMO has three foreign DNA regions such as gene expression adjustment region(Promoter), termination region (terminator) and structure gene. Detection of these regions can be done particularly by polymerase chain reaction (PCR). PCR-based detection can virtually be performed for any GMO within short of time. The most important prerequisite for the application of PCR-based detection is to decide abstraction method of efficient nucleic acids. Specially, in the case of processed food, because nucleic acids of foodstuffs are damaged by heat treatment (sterilization), pressure and fermentation, DNA must be extracted ken the samples prior to PCR analysis. Although many DNA extraction protocols are available, they have rarely been compared in a comprehensive method. In this study low widely used commercial and non-commercial DNA extraction methods-DNeasy$^{TM}$, Wizard$^{TM}$, CTAB, phenol/chloroform system-were compared with respect to the quality and yield of nucleic acids and insertion genes.nes.

• Parasites, Hosts and Diseases
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• v.53 no.2
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• pp.147-154
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• 2015

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (${\approx}375bp$) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ${\approx}550bp$ of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ${\approx}2$ oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.65 no.4
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• pp.496-507
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• 2020

Virus infections of apples result in lowered commercial qualities such as low sugar content, weakened tree vigor, and malformed fruits. An effective way to control viruses is to produce virus-free plants based on the development of an accurate and sensitive diagnostic method. In this study, real-time PCR assays based on SYBR Green I and TaqMan probes were developed for detecting ASGV, ASPV, and ApMV viruses. These methods can detect and quantify 10³ to 10¹¹ RNA copies/μL of each virus separately. Compared with methods with two different dyes, the SYBR Green I-based method was efficient for virus detection as well as for assay using the TaqMan probe. Field tests demonstrated that real-time PCR methods developed in this study were applicable to high-throughput diagnoses for virus research and plant quarantine.

• Korean Journal of Food Science and Technology
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• v.35 no.4
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• pp.591-597
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• 2003

To compare the quality of genomic DNA extracted from potato for PCR detection, four different methods, such as silica-based membrane method, silica-coated bead method, STE solution treatment, and CTAB-phenol/chloroform method, were evaluated. Also, to remove an excessive carbohydrate from the potato, ${\alpha}$- and ${\beta}$-amylase were used individually and in combination. When used both silica-based membrane method and silica-coated bead method combined with enzymes, the genomic DNAs were extracted from the raw potato with high purity for PCR. However, the silica-coated head method combined with enzyme treatment was the most efficient for extraction of the genomic DNA from the frozen fried potatoes. When applied with STE solution, the highly purified DNA was extracted from the raw potatoes without enzyme treatment in adequate yield for PCR. In cases of processed potatoes, such as frozen-fried potato and fabricated potato chips, CTAB-phenol/chloroform method is mostly feasible for DNA extraction and PCR efficacy at high sensitivity. As the results of PCR amplification, 216bp of PCR product was detected on 2% agarose gel electrophoresis, but any amplicons derived from New leaf and New leaf Y gene was not detected in any sample.