• Korean Journal of Agricultural Science
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• v.42 no.2
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• pp.99-103
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• 2015

Potato virus T (PVT) is a plant pathogen in the family Betaflexiviridae, group IV single-stranded positive sense RNA viruses. The major host of PVT is potato, and it has been reported in Ullucus tuberosus, Oxalis tuberosa and Tropaeolum tuberosum. This study aimed at developing reverse transcription (RT)-polymerase chain reaction (PCR) and nested PCR techniques for specific detection of PVT. Finally, Two RT-PCR primer sets were developed and verified. The RT-PCR products were amplified to 734 (PVT RT-PCR primer set 6) and 828 bp (PVT RT-PCR primer set 29) long to detect PVT. The nested PCR primer sets [PVT-N70/C20 ($734{\rightarrow}315bp$) and PVT-N75/C30 ($828{\rightarrow}529bp$)] were developed which are high sensitivity and verification for detection of PVT. Furthermore, a modified-positive control plasmid is use to verify contamination of laboratory in PVT detection. This study supported the diagnose PVT in potato or PVT related hosts.

• Food Science and Biotechnology
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• v.17 no.2
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• pp.357-361
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• 2008

One of the major allergenic proteins in common buckwheat (Fagopyrum elculentum) was found to be a BW10KD. In this work, allergenic BW10KD genomic DNAs from the native common buckwheat 'Pyeongchang' and Tartarian buckwheat 'Clfa47' were cloned by polymerase chain reaction (PCR), and their nucleotide sequences were determined. In addition, a novel PCR assay targeting the allergenic BW10KD gene was developed to detect and differentiate both buckwheat species in food. The nucleotide sequences of the BW10KD genomic DNA from 'Pyeongchang' and 'Clfa47' were 94% identical. Base differences in the nucleotide sequences of the BW10KD genes are probably useful as a molecular marker for species-specific identification. The 'Pyeongchang'-specific primer set 154PF/400PR and the 'Clfa47'-specific primer set 154DF/253DR generated 247 and 100 bp fragments in singleplex PCR, respectively. A duplex PCR assay with 2 species-specific primer sets simultaneously differentiated the 'Pyeongchang' and 'Clfa47' in a single reaction. The PCR assay also successfully allowed for the rapid detection of buckwheat ingredients in foods.

• BMB Reports
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• v.33 no.2
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• pp.162-165
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• 2000

Polymerase chain reaction (PCR) is well established as an indispensable tool of molecular biology; and yet a limitation for cloning applications continues to be that products often require subsequent restriction to be that products often require subsequent restriction digests, blunt-end ligation, or the use of special linear vectors. Here a rapid, PCR-based system is described for the simple, restriction enzyme-free generation of synthetic, 'restriction-like' DNA fragments with staggered ends. Any 3'- or 5'-protruding terminus, but also non-palindromic overhangs with an unrestricted single strand length are specifically created. With longer overhangs, "Restriction-PCR" does not even require a ligation step prior to transformation. Thereby the technique presents a powerful tool e.g. for a successive, authentic reconstitution of sub-fragments of long genes with no need to manipulate the sequence or to introduce restriction sites. Since restriction enzyme-free and thereby devoid the limitations of partial DNA digests, "Restriction-PCR" allows a straight one-step generation and cloning of difficult DNA fragments that internally carry additional sites for specific sequence insertions or deletions can be precisely engineered into genes of interest. With these properties "Restriction-PCR" has the potential to add significant speed and versatility to a wide variety of DNA cloning applications.

• Analytical Science and Technology
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• v.12 no.1
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• pp.80-83
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• 1999

Recently, it has been possible to the alphoid gene, which has X and Y specificity, and determine the sex from human physical evidence using PCR methods. Samples from single sources, PCR method applied to the alphoid gene results in highly sensitive and accurate results even when only 60 pg of the genomic DNA was available for sex determination. Even for samples containing DNA from more than one gender source where the female DNA was present in the amount 10 times than that of the male, sex determination was possible. Therefore, this result suggests that alphoid gene, which has X and Y specificity, could be used effectively for sex determination in case of mixed DNA samples from biological evidence.

• Journal of Korean Society of Environmental Engineers
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• v.29 no.8
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• pp.904-908
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• 2007

The protozoan pathogen Giardia lamblia has been major cause of waterborne enteric disease. In this study, we tried to identify G. lamlbia of human infectious species and to detect viable C. lamblia in river water samples including three sites of Han River mainstream and an its creek using PCR and RT-PCR technique. The PCR/RT-PCR methods were performed by using giardin primer based on the giardin gene targeting ventral disk of Giardia. Sensitivity testing in the DNA/RNA extraction and PCR/RT-PCR amplification steps showed that it was possible to detect a single cyst of G. lamblia and viable G. lamblia. The PCR/RT-PCR methods were compared with immunofluorescence(IF) assay by analyzing 48 samples collected from the mainstream water and the creek water. The mean concentration of the total cysts were 6.3 cysts/10 L(arithmetic mean, n = 48) and the positive detection rate were 62.5%(30/48). And the mean concentration of the cysts excluding empty cysts were 4.5 cysts/10 L and the positive detection rate were 52.1%(25/48). It resulted that 24 of 48 samples included Giardia lamblia by PCR assay and 10 of 48 samples included viable G. lamblia by RT-PCR assay. It resulted that the PCR/RT-PCR technique would be available to river water samples with low concentration of Giardia cysts. And it could support the Korean protozoan standard method, which provides useful information for species and viability.

• Korean Journal of Veterinary Research
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• v.41 no.4
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• pp.535-542
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• 2001

A multiplex PCR technique was developed for detecting specifically each Mycobacterium bovis, M. tuberculosis, M. avium and M. avium subsp, paratuberculosis, respectively, using clinical samples of field cattle. To apply this novel technique to clinical specimens, blood sample was obtained from live cows comprising 11 intradermal tuberculin test (ITT)-positive and 17 ITT-negative and tested by multiplex PCR. Positive results were obtained from 15 cows by the multiplex PCR, showing that 4 (23.5%) of the 17 ITT-negative cows were multiplex PCR positive. The multiplex PCR results also showed that among the 15 positive cows, 7 (46.7%) were infected with M. bovis, 1 (6.7%) with M. tuberculosis and 7 (46.7%) with M. avium. The sensitivity and specificity of multiplex PCR in comparison with those of ITT were 100% and 76.5%. The correlation between the multiplex PCR and ITT assays with blood samples was considered excellent, 85.7% agreement and ${\kappa}=0.72$. The results obtained, using reference mycobacterial strains and typed clinical samples, show that the multiplex PCR method may be a rapid, sensitive, and specific tool for the differential identification of various mycobacterial strains in a single-step assay. Therefore, multiplex PCR assay is a useful tool for early diagnosis of tuberculosis in live cattle and to identify the species or complex of mycobacterium from clinical samples.

• Journal of Physiology & Pathology in Korean Medicine
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• v.21 no.5
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• pp.1307-1312
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• 2007

This study was designed to investigate effectiveness of korean medicine therapy and the relation between effectiveness of that and single nucleotide gene polymorphism in stroke patients. This study was carried out on 92 stroke patients who were admitted to the department of acupuncture & moxibustion, college of Oriental medicine, Daegu Haany University and 112 healthy Korean. All patients were received Korean medicine therapy including acupuncture and herbal medicine for stroke and assessed by National Institutes of Health Stroke Scale(NIHSS). Blood samples from all subjects were obtained for DNA extraction. The extracted DNA was amplified by polymerase chain reaction(PCR). PCR products were visualized by 1.5% agarose gel electrophoresis. Through Pyrosequencing of PCR product, the polymorphism of single nucleotide gene was genotyped automatically. There were significant difference between before and after Korean medicine therapy in NIHSS. Genotypes were AA, AG, GG, but there was no significant difference between control and stroke groups. And there was not any statistical significant allelic frequency difference between control and stroke groups. We concluded that Korean medicine therapy in stroke patient can improve NIHSS, but there is no definite relation between effectiveness of Korean medicine therapy and single nucleotide gene polymorphism in stroke patients. This study need to be confirmed in large patients and further studies about relation with gene polymorphism are required.

• Journal of Plant Biology
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• v.38 no.3
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• pp.267-274
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• 1995

Based upon the nucleotide sequence of As strain of cucumber mosaic virus (CMV-As0 RNA4, coat protein (CP) gene was selected for the design of oligonucleotide primers of polymerase chain reaction (PCR) for detection and identification of the virus. Reverse transcription and polymerase chain reaction (RT-PCR) was performed with a set of 18-mer CMV CP-specific primers to amplify a 671 bp fragment from crude nucleic acid extracts of virus-infected leaf tissues as well as purified viral RNAs. The minimum concentrations of template viral RNA and crude nucleic acids from infected tobacco tissue required to detect the virus were 1.0 fg and 1:65,536 (w/v), respectively. No PCR product was obtained when potato virus Y-VN RNA or extracts of healthy plants were used as templates in RT-PCR using the same primers. The RT-PCR detected CMV-Y strain as well as CMV-As strain. Restriction analysis of the two individual PCR amplified DNA fragments from CMV-As and CMV-Y strains showed distinct polymorphic patterns. PCR product from CMV-As has a single recognition site for EcoRI and EcoRV, respectively, and the product from CMV-Y has no site for EcoRI or EcoRV but only one site for HindIII. The RT-PCR was able to detect the virus in the tissues of infected pepper, tomato and Chinese cabbage plants.

• Journal of Microbiology and Biotechnology
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• v.26 no.8
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• pp.1398-1403
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• 2016

The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10¹ copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10² copies/20 g fresh lettuce, 9.7 × 10³ copies/20 g frozen strawberries, and 4.1 × 10³ copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.

• Journal of Plant Biotechnology
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• v.45 no.2
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• pp.102-109
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• 2018

Thistle is a perennial plant that is widely used for medicinal purposes. Information on the genetic diversity of thistle populations are great important for their conservation and germ plasmic utilization. Although thistle is an important medicinal plant species registered in South Korea, no molecular markers are currently available to distinguish them from other similar species from different countries. In this study, we developed single nucleotide polymorphism (SNP) markers derived from the nuclear ribosomal DNA internal transcribed spacer (ITS) regions of genomic sequences to identify distinct Korean-specific thistle species via an amplification refractory mutation system (ARMS)-PCR and high resolution melting (HRM) curve analyses. We performed molecular authentication of four different kinds of thistle species from different regions using DNA sequences in the ITS intergenic region. We also developed a quantitative PCR assay using species-specific ITS primers, which allowed us to estimate the ratio of Korean-specific thistle species using varying ratios of mixed genomic DNA templates from the two species. The SNP markers developed in this study are useful for rapidly identifying specific thistle species from different countries.