• Horticultural Science & Technology
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• v.28 no.4
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• pp.672-677
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• 2010

A duplex PCR method was developed to detect a transformation vector pSPB130 used in the development of a genetically modified (GM) rose plant. To detect a GM rose plant, the anthocyanin synthase ($ANS$) was used as an endogenous reference gene of rose in PCR detection. The primer pair RHANS-KF/KR producing 107 bp amplicon was used to amplify the $ANS$ gene and no amplified product was observed in any of the 9 different plants used as a template. The primer pair GMRH-KF/KR was designed to amplify the junction sequence between 35S promoter and flavonoid 3',5'-hydroxylase ($F3^{\prime}5^{\prime}H$) gene in pSPB130. The detection limit of the duplex PCR method is approximately 0.5%. This result indicates that this duplex PCR method could be useful for monitoring unauthorized GM rose in Korea.

• Journal of fish pathology
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• v.5 no.2
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• pp.143-152
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• 1992

Metallothionein is an essential and common protein to regulate the intracellular concentration of heavy metals, which exist in most organisms from bacteria to vertebrates. Although the detailed function of metallothianein has not been fully identified until yet, it may be involoved in the cellular protection against the heavy metal toxicity and in the global regulation of several other genes and the expression of metalloproteins. We have cloned the full cDNA clone of metallothionein gene in Channel Catfish by Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR) starting from poly(A)-containing mRNAs. All PCR fragments have been subcloned into EcoRV site of pBluescript SK+ and dT-tailed at Smal site of pUC19, then PCR products are recovered by the double digestion of recombinant plasmids wiht EcoRI and HindIII, which are adjacent to EcoRV site in multicloning sites or by rapid PCR screening. The nucleotide sequence analysis of pMT150(one of the PCR clones) showed high homology with several other piscine metallothionein cDNA genes.

• Parasites, Hosts and Diseases
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• v.35 no.2
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• pp.111-118
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• 1997

Four separate pairs of oligonucleotide primers within the coding region in a T sergenti 33-kDa surface protein gene were selected to detect T. sergenti by PCR. The specificity of PCR-amplified DNA was examined by digestion with restriction enzyme 3nd Southern blot hybridization using T. sergenti p33 DNA probe. PCR appears to be specific for T. sergenti, without detectable signals from uninfected erythrocytes, uninfected bovine leukocytes and other hemoparasites, including A. morginnle and 3. ouata. Although 46 of 71 specimens (64.8%) from grazing cattle were microscopically positive. PCR in this study showed that 64 specimens (88.7%) were positive. Therefore, PCR proves a useful diagnostic tool for detecting T sergenti-infected cattle. In addition, it is also revealed that PCR was significantly more sensitive than traditional microscopic examination using Giemsa's stain.

• 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.

• The Plant Pathology Journal
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• v.16 no.1
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• pp.48-51
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• 2000

Citrus tristeza virus (CTV) was identified form CTV-infected early satsuma mandarin (Citus unshiu) and yuzu (C.junos) by RT-PCR. The total RNAs were isolated from citrus bark and seaf tissues infected with CTV and reverse transcription was followed with primers designed for amplifying CTV coat protein gene. DNA fragments 738 bp were amplified by RT-PCR and these products were colned for sequence analysis. Based on the sequence analysis, this PCR product has 97% sequence homology to CTV (T-385) CP gene isolated from USA. RT-PCR assay for CTV detection was more sensitivity than ELISA assay which was done with anti-CTV CP antibody. This is the frist report about CTV identification in Cheju island Korea.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2199-2205
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• 2016

Vibrio cholerae O1 serogroup Wave 3 El Tor strains are presently prevalent worldwide. The Wave 3 El Tor strains contain a TLC:RS1:CTX array on chromosome 1, and no element is integrated on chromosome 2. A multiplex PCR optimized to identify the TLC:RS1:CTX array of Wave 3 strains has been developed in this study. By using eight primers, the multiplex PCR can identify the characteristic CTX and RS1 array of Wave 3 strains from various arrays of strains belonging to other Waves. The four amplified DNA fragments of Wave 3 strains have been cloned in a vector, which could be used as a positive control for the multiplex PCR. This multiplex PCR and the positive control set could be useful tools for rapid recognition of Wave 3 El Tor strains.

• Korean Journal of Microbiology
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• v.37 no.1
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• pp.56-60
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• 2001

Molecular typing of Bacillus anthracis has been extremely difficult due to the lack of polymorphic DNA markers. Aiming to develop a DNA marker specific for Bacillus anthracis and to be able to discriminate this species from Bacillus genus, we applied the random amplified polymorphic DNA (RAPD)-PCR. We have identified B. anthracis from various Bacillus species. The analysis performed by RAPD clearly demonstrated substantial genetic variations among Bacillus species. This type of analysis is an easy, quick and highly discriminatory technique that may help in diagnosis of anthrax.

• Journal of Veterinary Clinics
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• v.37 no.1
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• pp.23-27
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• 2020

Between May and November 2018, babesiosis was examined in 162 bloods samples obtained to an animal hospital in Jeju island for anemia and medical examination. Sixty-two of 162 (38.3%) were positive by PCR. The ultra fast real-time PCR test with blood directly analyzed without DNA extraction showed the same results. Accurate diagnosis, treatment and prognosis of babesiosis should be combined with clinical symptoms, blood tests, the babesia antibody test, and the PCR antigen test. Ultra fast real-time PCR, with these tests, is expected to be a point-of-care testing (POCT) for easy, fast and accurate diagnosis of babesiosis in the veterinary clinic.

• Research in Plant Disease
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• v.13 no.3
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• pp.145-151
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• 2007

Xanthomonas axonopodis pv. glycines is the causal agent of bacterial pustule of soybean(Glycine max. (L.) Merr), which is one of the most prevalent bacterial diseases in Korea. In this study, Polymerase Chain Reaction (PCR) assay was applied to detect Xanthomonas axonopodis pv. glycines and to survey on seed contamination in 36 soybean cultivars of Korea. And we have to compare PCR assay with dilution-plating assay of detection and identification. We confirmed detection of pathogen from artificial infected seeds and natural Infected seeds using PCR assay. This assay gave results similar to a seed-wash dilution plating assay and proved more effective than classical methods. Results of survey on seed contamination by X. axonopodis pv. glycines from 36 cultivar seeds showed that the pathogen was detected from Pungsan-namulkong, Mallikong, Taekwangkong, Daemangkong, Ajukkarikong using PCR assay. Therefore, The PCR assay provides a sensitive, rapid tool for the specific detection of X. axonopodis pv. glycines in soybean seeds.

• Tuberculosis and Respiratory Diseases
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• v.43 no.6
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• pp.842-851
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• 1996

Background : Rifampicin(RFP) is a key component of the antituberculous shon-course chemotherapy and the RFP-resistance is a marker of multi-drug resistant(MDR) M. tuberculosis. rpoB gene encodes the ${\beta}$-subunit of RNA polymerase of M. tuberculosis which is the target of RFP. Recent reports show that rpoB gene mutations are the cause of RFP resistance of M. tuberculosis and the main mechanism of rpoB gene mutation is point mutation. And PCR-SSCP is a rapid and easy method for detecting point mutations. So we performed PCR-SSCP of rpoB gene of M. tuberculosis and compared the result with traditional RFP sensitivity test. Method : The 27 RFP sensitive M. tuberculosis culture isolates and 25 RFP resistant isolates were evaluated. The RFP sensitivity test was done at the Korean Tuberculosis istitute. The DNA was extracted by bead beater method and was amplified with primers TR-8 and TR-9 in a 20ul PCR reaction containing 0.1ul(luCi) [${\alpha}-^{32}P$] - dCTP. After amplification, SSCP was done using non-denaturaring polyacrylamide gel electrophoresis. Then direct sequencing was done in cases of different eletrophoretic mobility compared with that of H37Rv. In 19 cases, we compared PCR-SSCP results with patient's clinical course and the results of traditional RFP sensitivity test. Results : 1) All 27 RFP sensitive M. tuberculosis isolates showed the same electrophoretic mobility compared with that of H37Rv. And all 25 RFP resistant M. tuberculosis isolates showed different electrophoretic mobility. 2) The mechanism of rpoB gene mutation of M. tuberculosis is mainly point mutation. 3) The PCR-SSCP results correlate well with traditional RFP sensitivity and patient's clinical response to antituberculous treatment. Conclusion: The PCR-SSCP of rpoB gene is a very sensitive and rapid mehod in detecting RFP- resistant M. tuberculosis.