• Fisheries and Aquatic Sciences
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• v.4 no.1
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• pp.32-38
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• 2001

Several molecular biological techniques have been used to detect virus rapidly and accurately, but these methods have limitations in the early stage of viral infection with very low concentration of virus. We compared the detection limits of IMS-PCR, Western blot and ELISA with infectious hematopoietic necrosis virus OHNV). Four antibodies, rabbit anti-IHNV polyclonal antibody, anti-IHNV nucleocapsid protein monoclonal antibody, anti-IHNV nucleocapsid protein polyclonal antibody, and anti-IHNV glycoprotein polyclonal antibody, were tested to find out the most effective antibody for each method. The detection limit with IMS- PCR was $2\times10^6$ pfu when the viral RNA was extracted before RT-PCR. In the western blot with rabbit anti­IHNV polyclonal antibody one pfu of virus could be detected. In ELISA, 10 pfu of virus particles were detected with the same antibody.

• Biomedical Science Letters
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• v.27 no.4
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• pp.283-290
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• 2021

Severe acute respiratory syndrome coronavirus (SARS-CoV2) is a major coronavirus that infects humans with human Coronavirus (HuCoV)-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63, Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle east respiratory syndrome coronavirus (MERS-CoV). SARS-CoV2 is currently a global pandemic pathogen. In this study, we developed conventional RT-PCR based diagnostic system for the detection of SARS-CoV2 which is relatively inexpensive but has high stability and a wide range of users. Three conventional RT-PCR primer sets capable of forming specific band sizes by targeting the ORF1ab [232 nucleotide (nt)], E (200 nt) and N (288 nt) genes of SARS-CoV2 were developed, respectively, and it were confirmed to be about 10~100 times higher detection sensitivity than the previously reported methods. In addition, a standard positive control that can generate specific amplicons by reacting with the developed RT-PCR primers and verify the false-positiv from contamination of the laboratory was produced. Therefore, the diagnostic system that uses the RT-PCR method is expected to be used to detect SARS-CoV2.

• Food Science and Biotechnology
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• v.18 no.5
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• pp.1150-1154
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• 2009

Low levels of virus contamination and naturally occurring reverse transcription-polymerase chain reaction (RT-PCR) inhibitors restrain virus detection in oysters. A rapid and efficient oyster-processing procedure that can be used for sensitive virus detection in oysters was developed. Poliovirus type 1 Sabin strain was used to evaluate the efficacy of virus recovery. The procedure included (a) acid-adsorption and elution with buffers (0.25M glycine-0.14 M NaCl, pH 7.5; 0.25M threonine-0.14M NaCl, pH 7.5); (b) polyethylene glycol (PEG) precipitation; (c) resuspension in Tween 80/Tris solution and chloroform extraction; (d) the second PEG precipitation; (e) viral RNA extraction with TRIzol and isopropanol precipitation; and (f) RT-PCR combined with semi-nested PCR. The overall recovery of elution/concentration was 19.5% with poliovirus. The whole procedure usually takes 19 hr. The overall detection sensitivity was 4 RT-PCR units of genogroup I norovirus (NoV) and 6.4 RT-PCR units of genogroup II Nov/25 g of oysters initially seeded. The virus-detecting method developed in this study should facilitate the detection of low levels of NoV in oysters.

• Korean Journal Plant Pathology
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• v.14 no.6
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• pp.630-635
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• 1998

Tobacco mosaic tobamovirus (TMV), cucumber mosaic cucumovirus (CMV), cucumber green mottle mosaic tobamovirus (CGMMV) and zucchini yellow mosaic potyvirus (ZYMV) from individual fruits and seeds of hot pepper and cucumber were detected by the reverse transcription-polymerase chain reaction (RT-PCR). The dilution end-points for RT-PCR in curde sap from TMV. and CMV - infected hot pepper leaves and CMV - and CGMMV-infected cucumber leaves were 10-5. However, the amount of PCR product obtained from preparation of ZYMV-infected cucumber leaf was 10-fold lower than those of CMV or CGMMV-infected cucumber leaves. In hot pepper, both TMV and CMV were detected in all parts of the fruit wall tissue, but the yields of PCR products in the fruit stalk and its surrounding tissues were higher than those of the end parts of the fruit. On the other hand, in cucumber fruit infected with CMV, CGMMV or ZYMV, the fruit wall tissue and seed located in both stalk and end parts showed higher yields of PCR products than those of intermediate parts. Of five viruses that were analysed, only TMV in hot pepper seed, and CGMMV and CMV in cucumber seed were detected in testa parts.

• 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.

• 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.

• The Plant Pathology Journal
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• v.20 no.2
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• pp.142-146
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• 2004

Woody plant tissues contain great amounts of phenolic compounds and polysaccharides. These substances inhibit the activation of reverse transcriptase and/or Taq polymerase in RT-PCR. The commonly used multiple-step protocols using several additives to diminish polyphenolic compounds during nucleic acid extraction are time consuming and laborious. In this study, sodium sulfite was evaluated as an additive for nucleic acid extraction from woody plants and the efficiency of RT-PCR assay of commercial nucleic acid extraction kits and small-scale dsRNA extraction was compared. Sodium sulfite was used as an inhibitor against polyphenolic oxidases and its effects were compared in RNA extraction by commercial extraction kit and small-scale double-stranded RNA (dsRNA) extraction method for RT-PCR. During nucleic acid extraction, addition of 0.5%-1.5%(w/v) of sodium sulfite to lysis buffer or STE buffer resulted in lighter browning by oxidation than extracts without sodium sulfite and improved the RT-PCR detection. When commercial RNA extraction kit was used, optimal concentrations of sodium sulfite were variable according to the tested plant. However, with dsRNA as RT-PCR template, sodium sulfite 1.5% in STE buffer improved the detection efficiency of Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) in fruit trees, and reduced the unspecific amplifications signi-ficantly. Furthermore, when viruses existed at low titers in host plant, small-scale dsRNA extractions were very reliable.

• Korean Journal of Plant Tissue Culture
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• v.28 no.6
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• pp.335-339
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• 2001

Grapevine leafroll-associated 3 closterovirus (GLRaV-3) is phloem limited virus, and one of the most severe viral diseases found in Korea. However, nonhomogenous distribution and low concentration and seasonal variations of GLRaV-3 in grapevines remain as main problems which prevent the introduction and molecular biology or serology experiments. Virus-infected plantlet in vitro was obtained from node tissue cuttings, which was GLRaV-3 infected 'kyoho' vines. The amount of purified virus was highest in vitro plantlet. Moreover, viruses seem to be relatively homogeneously distributed in all organs including leaf, stem and callus derived from in vitro plantlets. RT-PCR detection using in vitro plantlet tissue as template was most effective. When comparing ELISA to RT-PCR, RT-PCR detection was 1,000 times as effective as ELISA. These results can be explained by improved quality such as tenderness or less tannins in plantlet in vitro. In conclusion, until infected herbaceous host will be available, tissue culture can be usefully adopted as a technique for a good source of GLRaV-3 closterovirus for further studies.

• The Plant Pathology Journal
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• v.39 no.4
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• pp.309-318
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• 2023

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, 'Candidatus Liberibacter asiaticus' (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, Diaphorina citri Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy nrdB gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the nrdB target as low as ~2.6 Log10 copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.

• Korean Journal of Veterinary Service
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• v.41 no.1
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• pp.29-39
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• 2018

In this study, we developed a sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid visual detection of foot-and-mouth disease virus (FMDV) circulated in Korea. The RT-LAMP was completed in 40 min at $62^{\circ}C$ and the results of the assay were directly detected by naked eye without any detection process. The assay specifically amplified all 7 serotypes of FMDV RNAs but not amplified other viral and cellular nucleic acids. The sensitivity of the RT-LAMP was $10^2$, $10^3$ and $10^3TCID_{50}/mL$ for serotype O, A and Asia 1 FMDV, respectively, which was comparable to conventional reverse transcription polymerase chain reaction (RT-PCR) and relatively lower than that of real time quantitative RT-PCR (qRT-PCR). Clinical evaluation of the RT-LAMP using different serotypes of Korean and foreign FMDV strains showed a 100% (35/35) agreement with the results of the RT-PCR and qRT-PCR. These results indicated that RT-LAMP assay developed in this study could be a valuable diagnostic method for FMDV monitoring and surveillance.