• Proceedings of the Korean Society of Veterinary Pathology Conference
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• 2002.11a
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• pp.139-139
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• 2002

돼지 설사유발 칼리시 바이러스(Porcine enteric calicivirus: PECV)도 자돈에서 설사를 일으키는 바이러스로 이미 알려졌다. RT-PCR과 nested PCR을 이용하여 국내 양돈장에서 PECV의 발생을 조사하고자 본 연구를 시도하였다. 설사 분변은 경기, 충남, 전북, 전남과 제주지역에 분포한 31개의 농장 102마리의 자돈에서 채취하여 의뢰된 것을 조사하였다. RT-PCR 과 nested PCR 을 위하여 RNA dependent RNA Polymerase (RDRP) 부위와 capsid 부위에서 각각 2 쌍의 primer를 작성하였다. RDRP 부위에서 RT-PCR을 시행했던 바, 3마리 (2.9％) 에서, nested PCR에서는 18마리 (17.6％)에서 양성반응이 나왔으며 capsid 부위에서 RT-PCR 결과 5마리 (4.9％), nested PCR에서는 18마리(17.6％)가 양성반응으로 확인되었다. 본 연구를 통하여 PECV가 국내에서 돼지 설사를 일으키는 주요 원인체 중 하나라는 것이 밝혀졌으며, nested PCR 기법이 돼지 설사분변에서 PECV를 검출하는 좋은 진단방법이었다.

• 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.

• Korean Journal of Plant Tissue Culture
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• v.25 no.6
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• pp.519-524
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• 1998

A simple and reliable method to diagnose cucumber green mottle mosaic virus of watermelon in Korea (CGMMV-WK) was determined by RT-PCR, and coat protein gene for CGMMV-WK was cloned. Comparing to a method reported by Lee et al. (1996), the method developed here showed a better RT-PCR reaction. RT-PCR was possible by one step in the PCR reaction mixture that contains 20 pmol of primer, reverse transcriptase (30 unit), RNasin (5 unit) using the crude RNA solution. RT-PCR condition for specifically diagnosing CGMMV-WK was that cDNA was synthesized at 42$^{\circ}C$ for 45 min followed by pre-denaturation at 95$^{\circ}C$ for 2 min, and then PCR reaction was carried out with a programmed condition that consisted of 36 sequential cycles at 96$^{\circ}C$ for 30 sec, 6$0^{\circ}C$ for 30 sec, and 72$^{\circ}C$ for 1 min. A gene encoding the coat protein of CGMMV-WK was cloned and characterized. Nucleotide sequence of coat protein gene of CGMMV-WK shared 98.77% and 99.38% of sequence identity with those of CGMMV-W and CGMMV-SH, respecitvely, however, all of amino acid sequences were same.

• 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 Veterinary Science
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• v.21 no.5
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• pp.71.1-71.10
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• 2020

Background: Feline calicivirus (FCV) is a major and highly infectious pathogen in cats worldwide. However, there have been limited studies about the status of FCV infections in Korea. Objectives: To investigate the current status of FCV infections in stray cats in Korea. Methods: A novel reverse transcription polymerase chain reaction (RT-PCR) assay was developed based on the conserved nucleotide sequences of reported FCV strains. Field swab samples were collected from 122 cats (2 hospital admitted cats and 120 stray cats) in 2016 and 2017. All the samples were tested by virus isolation and 2 different RT-PCRs, including the novel RT-PCR, for the detection of FCV. Results: The novel RT-PCR assay showed no cross-reactivity to the nucleic acids of the other feline pathogens tested, and the limit of detection was calculated as 10⁰ TCID₅₀/mL based on an in vitro assessment. The novel RT-PCR assay detected 5 positive samples from the 122 field samples, which showed perfect agreement with the results of the virus isolation method. In contrast, another RT-PCR assay used in a previous study in Korea detected no positive samples. The prevalence of FCV infection in stray cats was 2.5% (3/120) based on the results of virus isolation and the novel RT-PCR assays. Conclusions: The current study is the first report of the detection and prevalence of FCV in stray cats in Korea. The novel RT-PCR assay developed in this study showed high sensitivity and specificity, which indicates a useful diagnostic assay to identify FCV infection in cats.

• Research in Plant Disease
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• v.23 no.4
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• pp.363-366
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• 2017

Barley mild mosaic virus (BaMMV), Barley yellow mosaic virus (BaYMV) and Barley yellow dwarf virus (BYDV) have been identified as an important causative agents for an economically important disease of winter barley in Korea. In this study, a multiplex reverse transcription polymerase chain reaction (mRT-PCR) method was used for the simultaneous detection. Three sets of virus-specific primers targeted to the capsid protein coding genes of BaMMV, BaYMV and BYDV were used to amplify fragments that were 594 bp, 461 bp, and 290 bp, respectively. Several sets of primers for each target virus were evaluated for their sensitivity and specificity by multiplex RT-PCR. The optimum primer concentrations and RT-PCR conditions were determined for the multiplex RT-PCR. The mRT-PCR assay was found to be a better and rapid virus diagnostic tool of specific barley diseases and potential for investigating the epidemiology of these viral diseases.

• Biomedical Science Letters
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• v.22 no.4
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• pp.215-219
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• 2016

The problems of tuberculosis and its drug resistance are very severe. Therefore, rapid and accurate drug susceptibility assay is required. Recently, there has been an increased understanding of the genetic mechanism of Mycobacterium tuberculosis (MTB) drug resistance as well as advancement of molecular technologies. While many gene mutations correlate well with drug resistance, many genes do not show a strong correlation with drug resistance. For this reason, the current study assessed the utility of rpoB mRNA as a target to detect live mycobacteria. In this study, RT-PCR targeting of rpoB mRNA in BCG treated with rifampin was performed. Conventional RT-PCR and real-time PCR targeting rpoB mRNA as well as 85B mRNA was performed to determine whether these two methods could distinguish between viable and non-viable MTB. The levels of rpoB and 85B mRNA detected by RT- PCR were compared in parallel with colony forming unit counts of BCG that were treated with rifampin for different periods of time. The data suggests that that even though both mRNA levels of rpoB and 85B decreased gradually when rifampin-treatment increased, the rpoB mRNA seemed to represent live bacteria better than 85B mRNA. This study clearly indicates that RT-PCR is a good method to monitor viable cell counts in the liquid culture treated with the anti-tuberculosis drug.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.210-217
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• 2018

A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.

• Korean Journal of Ecology and Environment
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• v.37 no.1 s.106
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• pp.122-129
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• 2004

In an effort to develop the biomarker for monitoring the contamination of xenoestrogen in the freshwater environment of Korea, reverse transcription-polymerasechain reaction (RT-PCR) analysis of vitellogenin (VTG) gene expression was optimized in Hearisarsus Iaseo, Based on the homology of the VTG cDNA sequences between the common carp and zebra fish, a set of PCR primers for VTG mRNA amplification for H; labo was designed. VTG mRNA level in livers from female and male fishes was analyzed by RT-PCR following single injection of 17 beta estradiol($E_2$ 10 mg $kg^{-1}$ B.W.). As an internal control, beta actin mRNA was amplified. One us of total liver RNA was subjected to RT-PCR. In female the amount of PCR productof VfC gradually increased in the range from 16 to 34 cycles of amplification. On the contrary, in control male, PCR product first detected at 32 cycles of amplification and linearly increased up to 40 cycles of amplification. In $E_2$ injected male liver, the VTC mRNA level was similar to that in the female. Taken together, this result suggests that liver of male H. labo expresses minute amount of VTG mRNA which are2-l6 equivalent of female and that induction of VTG mRNA occurs in male liver after estrogen treatment. In conclusion, the optimized protocol for RT-PCR analysis of VTG mRNA expression in liver of male H. labo will provide the environmental monitoring method for the xenoestrogen contamination in the rivers in Korea.

• Journal of Plant Biotechnology
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• v.5 no.2
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• pp.83-86
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• 2003

A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two specific 22-mer primers located in coat protein gene of SPFMV. A 411 bp PCR-product was detected in virus infected plants as well as tissue culture raised sweet potato but not in healthy plants. For optimization of RT-PCR protocol, the optimum crude nucleic acid concentration, annealing temperature, primer concentration and numbers of PCR-cycle for maximum sensitivity and specificity were determined. The optimum condition for RT-PCR was as follows: RT-PCR reaction mixture was one-step mixture, containing 50 pmol of primer, 30 units of reverse transcriptase, 5 units of RNasin, and the crude nucleic acid extracts (200 ng). In RT-PCR, cDNA was synthesized at $42^{\circ}C$ for 45 min before a quick incubation on ice after pre-denaturation at $95^{\circ}C$ for 5 min. The PCR reaction was carried out for 40 cycles at $96^{\circ}C$ for 30 see, $63^{\circ}C$ for 30 sec, $72^{\circ}C$ for 1 min, and finally at $72^{\circ}C$ for 10 min. The viral origin of the amplified product was confirmed by sequencing, with the sequence obtained having $95-98\%$ homology with published sequence data for SPFMV. The benefits of this RT-PCR based detection of SPFMV would be simple, rapid and specific.