• Title/Summary/Keyword: RT-PCR analysis

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RT- PCR Analysis of Vitellogenin Gene Expression in Bombina orientalis (무당개구리 비텔로제닌 유전자의 발현의 RT- PCR 검출법)

  • 계명찬;이명식;강희정;정경아;안혜선
    • Korean Journal of Environmental Biology
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    • v.22 no.2
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    • pp.329-335
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    • 2004
  • To develop a biomarker for the monitoring of the contamination of estrogenic endocrine disrupters in the aquatic environment, reverse transcription -polymerase chain reaction (RT-PCR) analysis of vitellogenin (Vg) mRNA expression was optimized in Bombina orientalis, a Korean red bellied toad species. Based on partial cDNA sequences of both Vg and beta actin genes of B. orientalis, specific primers for RT-PCR of Vg and beta actin mRNAs were developed. Semiquantitative RT-PCR of the Vg mRNA in liver was optimized using a beta actin mRNA as an internal control in both sexes. In female RT-PCR using $1\;\mu{g}$ of the liver cDNA resulted in a linear increment in the PCR product of Vg from 18 to 34 cycles of amplification. In male, on the contrary, the RT- PCR product was first detected at 30 cycles of amplification and a linear increment was observed from 30 to 40 cycles of amplification, suggesting that male B. orientalis expresses minute amount of Vg mRNA which is a $2^{-12}$ equivalent of female. In conclusion, the optimized protocol for semiquantitative RT-PCR analysis of Vg mRNA level in B. orientalis male liver will be useful for the environmental monitoring the xenoestrogen contamination in the freshwater environment in Korea.

Identification and Differentiation of Cucumber Mosaic Virus Isolated from Forsythia koreana (CMV-Fk) Using PCR Techniques (PCR기법을 이용한 오이 모자이크 바이러스 개나리 분리주(CMV-Fk)의 동정과 구분)

  • 이상용;박선정;최장경
    • Korean Journal Plant Pathology
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    • v.14 no.4
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    • pp.308-313
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    • 1998
  • Reverse transcription and polymerase chain reaction (RT-PCR) techiniques were used to identification and differentiation of cucumber mosaic virus isolated from Forsythia koreana (CMV-Fk). RT-PCT used by two set of 20-mer primers one was CMV-common primers and another was CMV subgroup I-specific primers designed in a conserved region of the 3' end of CMV RNA3, amplified about 490 bp and 200 bp DNA fragments from CMV-Fk, respectively. CMV could be detected by RT-PCR at a dilution as low as 10-4 in forsythia crude sap extracts. Restriction enzyme analysis of RT-PCR products using EcoRI and MspI showed that CMV-Fk belonged to CMV subgroup I. But, analysis of RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP-PCR) showed heterogeneity of RNA3 between CMV-Fk and CMV-Y as a member of subgroup I.

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Detection of Cucumber Mosaic Virus by RT-PCR Using a Simple and Rapid Crude Sap Extraction Method (간이 조즙액 추출법을 이용한 RT-PCR 방법에 의한 오이 모자이크 바이러스의 검정)

  • 이상용;홍진성;이진상;최장경
    • Korean Journal Plant Pathology
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    • v.12 no.4
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    • pp.432-436
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    • 1996
  • 역전사 중합효소 연쇄반응(RT-PCR)을 이용하여 담배(Nicotiana glutinosa)에 증식시킨 7종의 오이 모자이크 바이러스(CMV)를 검정하였다. RT-PCR을 위한 간단하고 신속한 바이러스 핵산의 조즙액 추출법이 개발되었으며, CMV 외피단백질 유전자 부위를 기초로 하여 제작한 20개의 염기로 구성된 primer를 사용하여 RT-PCR을 실시한 결과, 약 490 염기쌍의 DNA 단편들이 이병식물의 조즙액으로부터 증폭되었다. EcoRI 및 MspI을 이용한 RT-PCR 산물의 분석에 의하여, 공시한 7종의 바이러스는 모두 CMV subgroup I으로 동정되었다. Ouchterlony 한천젤 이중 확산법을 이용한 항혈청 검정에서도 7종의 바이러스 모두 CMV-Y의 항혈청과 단일의 침강선을 형성하였다.

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Analysis of Vitellogenin Gene Expression by RT-PCR in Hemibarbus labeo (Cyprinidae) for the Analysis of Estrogenic Activity in Aquatic Environment (수환경 내 Estrogen 에스트로젠 활성 검출을 위한 누치 난황전구단백질 유전자 발현의 RT-PCR시험법)

  • Gye, Myung-Chan
    • 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.

RT-PCR Detection of Citrus Tristeza Virus form Early Satsuma Nandarin and Yuzu in Cheju Island

  • Kim, Daehyun;Jaewook Hyun;Hyunsik Hwang;Lee, Sukchan
    • 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.

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Application of Reverse Transcription Droplet Digital PCR for Detection and Quantification of Tomato Spotted Wilt Virus (Reverse Transcription Droplet Digital PCR을 활용한 Tomato Spotted Wilt Virus 검출 및 정량)

  • Lee, Hyo-Jeong;Park, Ki Beom;Han, Yeon Soo;Jeong, Rae-Dong
    • Research in Plant Disease
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    • v.27 no.3
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    • pp.120-127
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    • 2021
  • Plant viruses cause significant yield losses, continuously compromising crop production and thus representing a serious threat to global food security. Tomato spotted wilt virus (TSWV) is the most harmful plant virus that mainly infects horticultural crops and has a wide host range. Reverse-transcription quantitative real-time PCR (RT-qPCR) has been widely used for detecting TSWV with high sensitivity, but its application is limited owing to the lack of standardization. Therefore, in this study, a sensitive and accurate reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) method was established for TSWV detection. Additionally, we compared the sensitivities of RT-qPCR and RT-ddPCR for TSWV detection. Specificity analysis of RT-ddPCR for TSWV showed no amplification for main pepper viruses and negative control. TSWV transcripts levels measured by RT-ddPCR and RT-qPCR showed a high degree of linearity; however, the former yielded results that were at least 10-fold more sensitive and detected lower TSWV copy numbers than the latter. Collectively, our findings show that RT-ddPCR provides improved analytical sensitivity and specificity for TSWV detection, making it suitable for identifying low TSWV concentrations in field samples.

Rapid Detection and Identification of Cucumber Mosaic Virus by Reverse Transcription and Polymerase Chain Reaction (RT-PCR) and Restriction Analysis (역전사 중합효소련쇄반응(RT-PCR)과 제한효소 분석을 이용한 오이 모자이크 바이러스의 신속한 검정과 동정)

  • Park, Won Mok
    • 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.

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Quantitative Analysis of Feline Calicivirus Inactivation using Real-time RT-PCR (Real-time RT-PCR을 이용한 Feline Calicivirus 불활성화의 정량적 분석)

  • Jeong, Hye Mi;Kim, Kwang Yup
    • Journal of Food Hygiene and Safety
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    • v.29 no.1
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    • pp.31-39
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    • 2014
  • Norovirus causes acute gastroenteritis in all age groups and its food poisoning outbreaks are rapidly increasing in Korea. Reverse transcription-polymerase chain reaction (RT-PCR) is most widely used for the rapid detection of foodborne viruses due to high sensitivity. However, the false positive results of RT-PCR obtained against already inactivated viruses could be a serious drawbacks in food safety area. In this study, we investigated a method to yield true positive RT-PCR results only with alive viruses. To decompose the RNA genes from dead viruses, the enzymatic treatments composed of proteinse K and Ribonuclease A were applied to the sanitized and inactivated virus particles. Another aim of this study was to quantify the efficiencies of several major sanitizing treatments using real-time RT-PCR. Feline calicivirus (FCV) that belongs to the same Caliciviridae family with norovirus was used as a surrogate model for norovirus. The initial level of virus in control suspension was approximately $10^4$ PFU/mL. Most of inactivated viruses treated with the enzymatic treatment for 30 min at $37^{\circ}C$ were not detected in RT-PCR, Quantification results to verify the inactivation efficiencies of sanitizing treatments using real-time RT-PCR showed no false positive in most cases. We could successfully develope a numerical quantification process for the inactivated viruses after major sanitizing treatments using real-time RT-PCR. The results obtained in this study could provide a novel basis of rapid virus quantification in food safety area.

Characterization and RT-PCR Detection of Turnip Mosaic Virus Isolated from Chinese Cabbage in Korea (배추에서 분리한 순무 모자이크 바이러스의 특성 및 역전사 중합효소 연쇄반응법(RT-PCR)을 이용한 검정)

  • 박원목;최설란;김수중;최승국;류기현
    • Korean Journal Plant Pathology
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    • v.14 no.3
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    • pp.223-228
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    • 1998
  • Turnip mosaic virus)TuMV-Ca) was isolated from a Chinese cabbage showing severe mosaic and black necrotic spots symptoms in Korea. The virus was identified as a strain of TuMV by its host range test, particle morphology, serology, double stranded RNA analysis. For detection of the virus, reverse transcription and polymerase chain reaction(RT-PCR) was performed with a set of 18-mer TuMV-specific primers to amplify a 876 bp DNA fragment The virus was rapidly detected from total nucleic acids of virus infected tissues as well as native viral RNA of purified virion particles by RT-PCR. Detection limit of the viral RNA by RT-PCR was 10 fg.

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Development of a One-Step Duplex RT-PCR Method for the Simultaneous Detection of VP3/VP1 and VP1/P2B Regions of the Hepatitis A Virus

  • Kim, Mi-Ju;Lee, Shin-Young;Kim, Hyun-Joong;Lee, Jeong Su;Joo, In Sun;Kwak, Hyo Sun;Kim, Hae-Yeong
    • 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.