• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.881-884
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• 2000

A polymerase chain reaction (PCR) was performed to rapidly identify Lactobacillus plantarum from type strains and kimchi samples. The PCR experiments were carried out using specific oligonucleotide primer sets based on the 16S rRNA gene sequences of L. plantarum. The expected DNA amplificate of 419 bp was obtained when either purified DNA or whole cells of L. plantarum strains reacted with LP primers, yet not with any of the other strains. The PCR product was confirmed by DNA sequencing. Accordingly, since the PCR method used is simple, specific, and rapid, it will be useful for monitoring and evaluation L. plantarum in the mixed microbial population found in kimchi.

• Korean Journal of Microbiology
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• v.40 no.3
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• pp.254-256
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• 2004

Polymerase chain reaction (PCR) is a powerful tool for precisely amplifying selected DNA sequences that have had a broad impact on genomic studies. When examining human $\alpha$- and $\beta$- tryptase genes which have 95% DNA homology, inconsistent PCR amplification of genomic sequences hampered our progress. This study suggests that long PCR technique on the original DNA digested with restriction enzymes improves both efficiency and sensitivity of PCR. These improved results seem to derived from the effective denaturation of the original genomic DNA template or reduction of formation of secondary structures that block either primer annealing or extension in PCR. Elimination of homo- or hetero-duplex products derived from highly homologous genes provides an additional advantage in this study. This communication describes how the use of restriction enzymes improved these efficiencies, and also facilitated studies of highly homologous genes including tryptase genes.

• BMB Reports
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• v.33 no.6
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• pp.537-540
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• 2000

A simplified but reliable method was developed for the polymerase chain reaction (PCR)-based detection of genetically modified (GM) plants. The modified CTAB (mCTAB) method enabled us to prepare a high quality of genomic DNA from several hundred plant leaf samples in one day. Using DNA samples prepared from seven dicots and two monocots, approximately 1.75-kb regions spanning 17 S to 25 S ribosomal RNA genes were successfully amplified in a 2X PCR pre-mix containing BLOTTO. Further fidelity assessment of the mCTAB method by PCR analysis with Roundup Ready soybean (RRS) and non-RRS plants showed that the DNA samples prepared alternately from each of two lines were evidently free of cross-contamination. These results demonstrate that the mCTAB method is highly recommended for the rapid detection of transgenes in large numbers of leaf samples from diverse transgenic plants.

• 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 Environment and Ecology
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• v.24 no.3
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• pp.318-324
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• 2010

The aim of this study is to examine feeding habits of the Korean water deer(Hydropotes inermis argyropus) from its rumen contents using a PCR-DGGE method. For this study, rumen contents were collected from water deer causalities by natural death or road-kill in two different sites(Cheorwon, Gangwon province and the Eastern part of Jeonnam province). DNA was extracted from rumen contents of a total of 44 individuals. Two primers, rbcLZ1aF(GC) and rbcL19bR, were used for PCR amplifications of ribulose-1,5-bisphosphate carboxylase large subunit (rbcL) gene. Among 44 samples, twenty-nine samples were successfully amplified by PCRs. The 29 PCR products of partial rbcL gene were applied for PCR-DGGE. Totally, six families of plants were detected from the diet analyses. Five families of plants were found in Cheorwon, Gangwon province, but only three families of plants were found in the Eastern part of Jeonnam province. The PCR-DGGE method will provide us with a potential tool to study feeding habits of ungulates including water deer, even though our results failed to identify the prey plants at the level of species.

• Journal of Life Science
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• v.18 no.3
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• pp.374-380
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• 2008

Rapid detection of enterovirus (EVs) is important in the management of aseptic meningitis. We examined the relative efficiency and specificity of the real-time nucleic acid sequence-based amplification (NASBA) comparing with the established reverse transcription polymerase chain reaction (RT-PCR) and viral culture method which were used for the detection of enterovirus RNA in clinical specimens. Of the total 292 samples, 145 were found to be positive to enterovirus RNA by real-time NASBA, 101 were positive by viral culture, and 86 were positive by RT-PCR. 147 samples and 46 samples were determined to be negative and positive by all methods respectively, but 4 samples were positive only by real-time NASBA. To compare the specificity of each method, various clinical samples which were diagnosed for herpes simplex virus (HSV)-1, HSV-2, adenovirus, mumps, and rhinovirus were applied. Except one rhinovirus sample which was false positive to enterovirus RNA by RT-PCR, the other different samples were negative to all three methods. The real-time NASBA procedure can be completed within 5 hours in contrast with 9 hours for the RT-PCR and 3-14 days for the viral culture. From this study, it was suggested that the real-time NASBA assay could be a standardized, rapid, specific, and sensitive procedure for the detection of enterovirus RNA.

• 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.

• Research in Plant Disease
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• v.18 no.2
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• pp.73-79
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• 2012

Phytophthora katsurae is a fungal pathogen responsible for chestnut ink disease. We designed two duplex primer sets (SOPC 1F/1R+KatI 3F/5R, SOPC 1-1F/1-1R+KatI 3F/5R) to detect P. katsurae. SOPC 1F/1R and SOPC 1-1F/1-1R primer pairs were designed for sequence characteristic amplification regions (SCAR) marker, and KatI 3F/5R primer pair was used for P. katsurae-specific primer designed from internal transcribed spacer (ITS) region. To assess the sensitivity of duplex PCR, genomic DNA was serially diluted 10-fold to make the final concentrations from 1 mg/ml to 1 ng/ml. The sensitivity for two primer sets were 1 ${\mu}g/ml$ and 100 ng/ml, respectively. To find detection limits for zoospores of P. katsurae, each zoospore suspension was serially diluted 10-fold to make the final concentrations from $1{\times}10^6$ to $1{\times}10^2$ cells/ml, and then DNA was extracted. The limits of detection for all of two primer sets were $1{\times}10^5$ cells/ml. All of two primer sets were specific to P. katsurae in PCR detection and did not produce any P. katsurae-specific PCR amplicons from other 16 Phytophthora species used as the control. This study shows that duplex PCR using two primer sets might be a useful tool for rapid and efficient detection of P. katsurae.

• 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 Clinical Laboratory Science
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• v.53 no.1
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• pp.49-59
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• 2021

This study investigated the use of real-time PCR melting curves for the diagnosis of blaKPC and blaNDM genes among the most frequently detected carbapenemase-producing Enterobacteriaceae in Korea. As a means of addressing the shortcomings of phenotype tests and conventional PCR. The modified Hodge test confirmed positivity in 25 of 35 strains, and carbapenemase inhibition testing confirmed positivity in 14 strains by meropenem+PBA or meropenem+EDTA. PCR analysis showed amplification products in 25 strains of Klebsiella pneumoniae carbapenemases (KPC), 10 of K. pneumoniae, 5 of E. coli, 5 of A. baumannii, 4 of P. aeruginosa, and 1 of P. putida. New Delhi metallo β-lactamase (NDM) identified amplification products in 8 strains, that is, 2 K. pneumoniae, 3 E. coli, 1 P. aeruginosa, 1 E. cloacae, and 1 P. retgeri strains. Real-time PCR melting curve analysis confirmed amplification in 25 strains of KPC and 8 strains of NDM, and these results were 100% consistent with PCR results. In conclusion, our findings suggest early diagnosis of carbapenem resistant Enterobacteriaceae by real-time PCR offers a potential means of antibacterial management that can prevent and control nosocomial infection spread.