• Journal of Food Hygiene and Safety
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• v.23 no.2
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• pp.121-128
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• 2008

The aim of this study was to develop a LightCycler-based real time PCR (LC-PCR) assay and to evaluate its diagnostic use for the detection of Staphylococcus aureus in raw milk samples. Following amplification of 113 bp of coa gene encoding an coagulase precursor specific for Staphylococcus aureus, melting curve and DNA sequencing analysis was performed to verify the specificity of the PCR products. Amplification of 209 bp gene encoding an altered penicillin-binding protein, PBP2a (mecA), melting curve analysis and DNA sequencing analysis was performed to verify methicillin resistance Staphylococcus aureus (MRSA). According to this study, 6 of 647 raw milk samples showed S. aureus positive and 2 of them showed a mecA positive and the detection limit was 10 fg of DNA. And we also isolated Staphylococcus chromogenes a causative agent of exudative epidermitis in pigs and cattle from 3 samples.

• Journal of fish pathology
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• v.10 no.1
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• pp.39-44
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• 1997

Metallothionein is an important and essential protein to control the intracellular concentration of heavy metals, which exist in all organisms from bacteria to vertebrates. Although the detailed functions and induction mechanisms of metallothionein gene have not been clearly characterized until yet, the structure of several metallothionein genes has been revealed. Especially, piscine metallothionein is regarded as an important protein because it is induced by several heavy metal pollutants and environmental stress and it could be determined the comparative amount of heavy metals and the extent of environmental stress by assaying the RNA transcript of metallothionein gene in the method of the quantitative RT-PCR(Reverse Transcriptase Polymerase Chain Reaction). In this study I have characterized the 450 bp PCR fragment of metallothionein gene amplified by using the mixture of internal specific primers and universal 3' end primer. The nucleotide sequence analysis of 450 bp PCR fragment amplified in cDNA library of channel catfish did not show strong homology to other piscine metallothionein genes.

• 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.

• The Korean Journal of Mycology
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• v.41 no.1
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• pp.52-55
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• 2013

Phoma glomerata (Corda) Wollenw. & Hochapfel is a pathogenic fungus causing spot diseases of plant leaves and fruits. This fungus is important in plant quarantine of seedlings and fruits in Korea. The aim of this study was to develop a sensitive and effective diagnostic method for P. glomerata detection in imported plants. The fungal species-specific PCR primers were designed based on the nucleotide sequences of the translation elongation factor 1 alpha gene and their specificity and sensitivity were tested. The designed primers named as PhoGlo-F and PhoGlo-R amplified specifically a 170 bp sized DNA band of the target gene from the genomic DNA of P. glomerata. No amplicon was produced from genomic DNAs of 16 other Phoma spp. and reference fungal species tested. Moreover, PhoGlo-F/PhoGlo-R primers successfully worked with real-time PCR technique. The detection limit of DNA content by conventional and real-time PCR were 10 pg and 1pg of the genomic DNA of P. glomerata, respectively. We believed that the developed makers would be very useful for P. glomerata detection.

• 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.

• Research in Plant Disease
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• v.21 no.4
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• pp.321-325
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• 2015

A rapid and simple RT-PCR diagnosis method for detection of Rice stripe virus (RSV), one of major virus infecting rice, was developed using porous ceramic cubes in this study. The porous ceramic cube can rapidly absorb biological molecules such as small-sized proteins and nucleic acid fragments into its pores. We examined whether this ability of porous ceramic cubes could be applied for isolating viral nucleic acids or particles from the RSV- infected plant tissues. In this study, we found that the porous ceramic cube was capable of absorbing a detection level of viruses from the rice tissues infected with RSV and established RT-PCR-based RNA diagnosis method using porous ceramic cubes.

• Parasites, Hosts and Diseases
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• v.50 no.2
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• pp.157-159
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• 2012

The aim of this study was to assess the usefulness of PCR for diagnosis of Trichomonas vaginalis infection among male patients with chronic recurrent prostatitis and urethritis. Between June 2001 and December 2003, a total of 33 patients visited the Department of Urology, Hanyang University Guri Hospital and were examined for T. vaginalis infection by PCR and culture in TYM medium. For the PCR, we used primers based on a repetitive sequence cloned from T. vaginalis (TV-E650). Voided bladder urine (VB1 and VB3) was sampled from 33 men with symptoms of lower urinary tract infection (urethral charge, residual urine sensation, and frequency). Culture failed to detect any T. vaginalis infection whereas PCR identified 7 cases of trichomoniasis (21.2%). Five of the 7 cases had been diagnosed with prostatitis and 2 with urethritis. PCR for the 5 prostatitis cases yielded a positive 330 bp band from bothVB1 and VB3, whereas positive results were only obtained from VB1 for the 2 urethritis patients. We showed that the PCR method could detect T. vaginalis when there was only 1 T. vaginalis cell per PCR mixture. Our results strongly support the usefulness of PCR on urine samples for detecting T. vaginalis in chronic prostatitis and urethritis patients.

• Research in Plant Disease
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• v.13 no.3
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• pp.137-144
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• 2007

Fifty nine strains of Agrobacterium vitis, the causal agent of crown-gall disease on grapevine, originating from different geographical regions and 16 grapevine cultivars including 35 Kyoho cultivar of Korea, were characterized by PCR polymorphic analysis using Universal Rice Primer(URP). Of 12 URP primers, primers URP1F, URP2R, URP2F, and URP4R, URP17R were available for detecting PCR polymorphic bands among the A. vitis strains. PCR polymorphic bands produced by primers URP2F and URP17R were profiled to 12 strain types. A. vitis strains originated from Kyoho cultivar of grapevine showed relatively simple genetic diversify of the four PCR types, while the A. vitis strains originated from other grapevine cultivars and type culture strains showed various genetic diversity with 8 types. Unweighted Pair-Group Method with Arithmetic mean(UPGMA) cluster analysis using the URP-PCR polymorphic bands showed 59.4. vitis strains are genetically clustered into large seven groups.

• Korean Journal of Veterinary Research
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• v.42 no.2
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• pp.225-229
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• 2002

We report that mycoplasma organisms from lung tissues of slaughter pigs were identified to genes fragments with references use of nested-PCR technique(nPCR). Seven strains of mycoplasma species were isolated from 70 lung tissues. The organisms were detected by in vitro amplification of 16S rRNA and 23S rRNA genes. Nucleotide sequences of the spacer between 16S and 23S in the ribosomal RNA operons of mycoplasma were identified by the analysis of products from the nested PCR. Four common PCR primers, MhF1, MhF2 MhR1 and MhR2, were designed by analysis between these sequences by first amplified with F1, R1 and second with F2, R2, respectively. Specific amplification of the spacer region for reference strains of M. hyopneumoniae, M. hyorhinis, M. flocculare were confirmed by first round of PCR in which the traduced fragments of 690bp, 460bp, 630bp. But amplications of second round was changed to 240bp, 210bp, 230bp, respectively. Three different strains (M. hyopneumoniae:4, M. hyorhinis:2, M. flocculare:1) were detected by the nested-PCR technique. The results suggest that the detection of swine mycoplasma by n-PCR can be analyzed the nucleotide sequences between rRNA operons and homology study.