• 대한수의학회지
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• 제39권1호
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• pp.104-110
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• 1999

Field infectious bursal disease viruses (IBDVs) were isolated from IBDV-suspected commercial chickens. The variable region in VP2 gene of six Korean IBDV isolates (K-IBDVs) and IBD vaccines was examined using the reverse transcriptase / polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) assay. With all K-IBDVs and vaccine IBDVs, a 474-bp fragment of the VP2 gene was amplified and tested with various restriction enzymes. Restriction enzymes BstNI and StyI differentiated K-IBDV isolates and IBD vaccines into four groups. Restriction enzyme profiles of K-IBDV isolates were different from them of IBD vaccines. K-IBDV isolates except for 310 isolate had specific SspI and TaqI recognition sites, which were recognized in highly virulent IBDVs, but IBD vaccines had no those sites. This study showed that RT/PCR-RFLP assay was thought to be valuable tool for differentiation of IBDVs and identification of highly virulent IBDV.

• 대한수의학회지
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• 제36권3호
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• pp.627-633
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• 1996

To analyze the genomic diversity of transmissible gastroenteritis virus (TGEV), the N-terminal half of the spike (S) glycoprotein gene and nonstructural protein gene (open reading frames 3 and 3-1) were amplified by reverse transcriptase reaction and polymerase chain reaction (RT-PCR), and analyzed using restriction fragment length polymorphism (RFLP) patterns of the amplified DNA. In this study, TGEV Miller (M6) and Purdue (P115) strains were used as reference strains, and two vaccine strains (MSV and STC3) and four Korea isolates (P44, VRI-WP, VRI-41, and VRI-48) were analyzed. All TGEV strains were amplified with three TGEV primer pairs. Although there was some exception in RFLP analysis, this method differentiated TGEV strains into following groups : Miller group (M6 and MSV), Purdue group (PUS, STC3, P44, VRI-WP, VRI-41, and VRI-48). Using Sau3AI and SspI, VRI-48 was differentiated from the Miller and Purdue type viruses. The RT/PCR in conjuction with RFLP analysis was a rapid and valuable tool for differentiating several strains of TGEV. This study revealed the occurences of distinct difference in genome of TGEV strains.

• The Plant Pathology Journal
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• 제18권1호
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• pp.12-17
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• 2002

For the molecular detection of Sweet potaio feathery mottle virus (SPFMV) from diseased sweet potato plants, reverse transcription and polymerase chain reaction (RT-PCR) was performed with the use of a set of virus-specific primers to amplify an 816 bp product. The viral coat protein gene was selected for the design of the primers. No PCR product was amplified when Turnip mosaic virus, Potato vims Y or Cucumber mosaic virus were used as template in RT-PCR with the SPFMV-specific primers. The lowest concentration of template viral RNA required for detection was 10 fg. The vim was rapidly detected from total nucleic acids of leaves and roots from the virus-infected sweet potato plants as well as from the purified viral RNA by the RT-PCR. Twenty-four sweet potato samples were selected and analyzed by RT-PCR and restriction fragment length polymorphism (RFLP). RFLP analysis of the PCR products showed three restriction patterns, which resulted in some point mutations suggesting the existence of quasi-species for the vims in the infected sweet potato plants.

• 대한바이러스학회지
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• 제27권2호
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• pp.169-176
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• 1997

In this study, the feasibility of identification and genotypic differentiation of enteroviruses was investigated by using nested reverse transcription-polymerase chain reaction (nested RT-PCR), single-stranded conformation polymorphism (SSCP), and restriction fragment length polymorphism (RFLP) techniques. Two hundred seventy-four clinical samples were assayed by both nested RT-PCR and tube culture method using MRC-5 and MK cells; 58 (86.6%) out of 67 enterovirus culture-positive samples contained enteroviral RNA. In addition, 114 (55.1%) of 207 samples from patients with suspected enteroviral CNS disease with negative viral cultures were positive by the nested RT-PCR. The nested RT-PCR products were genotyped by the SSCP method and the results were compared with serotypes. We could differentiate 6 subtypes, 3 of which are similar to coxsackievirus B3, B5, echovirus 11, plus 3 other subtypes. RFLP cleaved with Sty I, Bgl I, and Xmn I yielded characteristic patterns for each laboratory strains. This study demonstrates the usefulness of the RT-PCR for the rapid diagnosis of enterovirus infection and the potentials of the SSCP method for differentiation of enterovirus strains.

• 대한바이러스학회지
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• 제28권2호
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• pp.165-174
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• 1998

Characterizations of the VP4 (P type) and VP7 (G type) genes of Korean isolates of bovine rotavirus were performed using RT-PCR/RFLP and nucleotide sequencing analysis. After RT-PCR amplification of partial length (1094bp) of the VP4 and full length (1062bp) of the VP7 genes, amplified PCR products were digested with restriction endonucleases and digestion patterns were compared with those of reference rotaviruses. With the VP4 genes, four RFLP (A-D) profiles were observed; three (A, Band C) were the same as those of bovine rotavirus NCDV (P[1]), IND (P[5]) and B223 (P[11]), respectively. Profile D was the same as that of porcine rotavirus OSU (P[7]). With the VP7 genes, five RFLP profiles (I-V) were observed; three of them (I, II and III) were the same as those of bovine rotavirus NCDV (G6), Cody 1-801 (G8), and B223 (G10), respectively. Profile IV and V were atypical to those of reference bovine rotaviruses used in this study. These two profiles were identified as G6 and G5, respectively, after analyzing and comparing the nucleotide sequences. The G typing analysis revealed that 61.9% (26/42) were G6, which included G6 subtype; 28.6% (12/42) were G5; 7.1% (3/42) were G10; 2.4% (1/42) were G8. The P typing analysis revealed that 54.8% (23/42) were P[5]; 28.6% (12/42) were P[7]; 11.8% (5/42) were [11]; 4.8% (2/42) were P[1]. Our results showed that G6/P[5] were the most prevalent rotaviruses in diarrheic calves in Korea. Also, this is the first report that G5/P[7] rotaviruses were identified from cattle with diarrhea.

• 대한수의학회지
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• 제38권2호
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• pp.304-313
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• 1998

Sixteen Korean field transmissible gastroenteritis viruses (TGEVs) were isolated using swine testicular cell (STC) and the genomic diversity of them was analyzed. All TGEV isolates produced a typical cytopathic effect in STC and were confirmed as TGEV by immunofluorescence assay using monoclonal antibody against TGEV and PCR using TGEV specific primers. RNAs from TGEV field isolates and vaccine TGEV were extracted and amplified by RT and PCR. The RT-PCR products were digested with selected restriction enzymes and analyzed RFLP patterns. The N-terminal end region of S gene and ORF 3 and 3-1 genes of TGEV amplified by TGEV specific primer pairs seemed to be conserved. Most specific variations were detected in S gene amplified by TGEV 4/6 primer pairs which includes antigenic sites A and D. When the PCR products were treated with Sau3AI and Ssp I, Bvac(vaccine strain), field isolates 133 and 347 were differentiated from Miller and Purdue types. In the case of D5 field isolates, it was classified into Purdue type by Sau 3AI but classified into independent TGEV by Ssp I. Two different TGEV strains from D2 sample were confirmed by plaque purification and RT-PCR-RFLP analysis. To investigate the change occurring in TGEV genome after serial passage, the TGEV P44 strain was passaged through STC. There were specific changes in S gene and a large deletion was observed in ORF 3 and 3-1 genes. These studies showed that a distinct difference in genome exists among TGEV field isolates.

• 식물병연구
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• 제17권2호
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• pp.211-215
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• 2011

모자이크증상을 나타내는 열무로부터 Cucumber mosaic virus의 한 계통을 분리하고 특성을 조사하였다. Vigna unguiculata, Chenopodium amaranticolor and Gomphrena globosa에서 분리 바이러스의 기주반응과 dsRNA 분석, RT-PCR 검정, PCR-RFLP, 혈청학적 분석을 통하여 CMV의 한 계통임을 확인하였다. 이 CMV 계통을 다양한 지표식물에 접종하였을 때, Nicotiana benthamiana와 N. glutinosa, N. tabacum, 고추, 오이 그리고 멜론에서는 전형적인 강한 모자이크 병징이 나타났으나, 열무, 배추, 적갓은 매우 약한 병징을 나타내는 특징을 보였다. 새롭게 분리된 CMV 계통은 가지과, 박과 및 배추과 등 광범위한 작물에 감염성을 나타내었으며, 배추과에서의 감염성 차이를 근거로 Gn-CMV로 명명하였다. Double-stranded (ds) RNA를 분리한 분석에서 Gn-CMV는 기 보고된 CMV 계통과 마찬가지로 3.3, 3.0, 그리고 2.2 kb의 독립된 게놈으로 구성되어 있었으며, SDS-PAGE와 Western blotting 분석으로 통하여 28 kDa의 외피단백질을 확인할 수 있었다. RT-PCR로 얻어진 증폭산물을 Cac8I, ClaI and MspI을 이용한 PCR-RFLP를 통하여 CMV subgroup I 임을 확인할 수 있었다.

• 식물병연구
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• 제14권3호
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• pp.229-232
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• 2008

모자이크 증상의 과꽃(Callistephus chinensis L.)으로부터 Cucumber mosaic virus (CMV)의 한 계통(Cas-CMV)를 분리하고, Fny-CMV와 As-CMV를 대조로 기주반응, dsRNA, RT-PCR 및 RFLP분석을 통하여 바이러스를 동정하였다. Cas-CMV의 특징적인 기주반응의 차이는 박과 식물에서 발현되는 강한 병정이었으며, 특히 쥬키니호박에 접종하였을 때에는 접종 15-20일 후에 심한 모자이크 증상과 함께 어린 식물이 고사되는 괴저현상을 나타냈다. DsRNA분석과 RT-PCR실험의 결과는 Cas-CMV가 서브그룹 I의 CMV에 속하는 것으로 나타났으며, 더욱이 HindIII를 이용한 RFLP 분석은 Cas-CMV가 서브그룹 IA 구분되었다.

• Tuberculosis and Respiratory Diseases
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• 제62권5호
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• pp.406-416
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• 2007

연구배경: 단일염기다형성(Single nucleotide polymorphism, SNP)은 인간의 유전자 서열 1000염기에 1개 빈도로 발견되어 인간은 대략 300만개의 유전자 다형성을 가지고 있다. 이 유전자 다형성의 조합결과로 인간의 개체 간 특성들이 결정되는 것으로 이해되고 있다. 이러한 다형성들의 조합양상에 따라 특이 질환에 대한 유전자 감수성 또한 달라지게 되므로 최근에는 많은 질환들과 유전자 다형성들과의 상관관계를 보는 연구들도 활발하게 진행되고 있다. 이러한 SNP분석은 큰 집단을 대상으로 진행되어 지므로 적은 비용으로 정확하게 그리고 대용량으로 분석할 수 있는 방법이 필요하다. 방 법: 대상 환자 89명의 genomic DNA를 가지고서 promotor상에 위치한 -37과 -524 염기부위에서 유전자 다형성을 보이는 것으로 보고되어져 있는 RRM1(ribonucleotide reductase M1) 유전자를 대상으로 PCR-RFLP(polymerase chain reaction-restriction fragment length polymorphism)와 real-time PCR(RTPCR, TaqMan probe assay)을 동시에 시행한 후 각각의 결과를 비교 분석하였다. 결 과: 대상 DNA 89예 중 -37에서는 2예(2.17%), -524에서는 15예(16.26%)가 서로 다른 양상을 보였다. 결과 차이를 보인 샘플 17예를 대상으로 직접 염기서열 분석을 시행하여 본 결과, 17예 모두 RT-PCR에서 확인되었던 결과와 일치함을 확인할 수 있었다. 추가 샘플 138예를 대상으로 RT-PCR을 2회 연속 실행하여 genotyping을 해 본 결과 98%이상의 높은 일치율을 보였으며, 그중 10예를 무작위로 골라 직접 염기서열 분석을 시행하여 본 결과, 역시 100%일치, 높은 정확도를 보였고 이는 in-tube assay 방식으로 샘플의 오염을 최소화 할 수 있었으며 72 well based system(Corbett Research)을 이용함으로 1회 유전자 증폭반응을 통해 많은 검체를 한 번에 확인할 수 있어 매우 빠른 검사방법 이었다. 결 론: 큰 집단을 대상으로 다량의 SNP를 분석하기 위한 실험 방법으로는 RT-PCR이 신속하면서도 정확한 결과를 얻을 수 있는 방법으로 사료된다.

• 식물병연구
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• 제14권1호
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• pp.71-75
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• 2008

전형적인 모자이크 증상을 나타낸 토끼풀로부터 Peanut stunt virus(PSV)를 분리하여 Tr-PSV로 명명하였다. 이 연구에서는 Tr-PSV의 특성을 기주반응, 혈청학적 성질, dsRNA분석, RT-PCR 및 RFLP 분석 등을 통하여 지금까지 잘 알려진 ER-PSV Fny-CMV 및 LS-CMV와 비교하였다. Tr-PSV는 접종한 Nicotiana속 식물에서는 모두 전신감염되었으며, C. amaranticolor에서는 접종엽에 국부병반을 형성하였다. 그러나 동부에서는 대조로 이용한 ER-PSV와 마찬가지로 전신감염되어, 접종엽에 국부병반을 형성하는 대조의 CMV계통과는 구분되었다. Tr-PSV에 감염된 N. benthamiana 잎으로부터 추출한 dsRNA는 대조의 PSV나 CMV와 유사한 분자 크기의 4종의 dsRNA가 확인되었고, satellite RNA의 존재는 인정되지 않았다. Cucumovirus 특이적 프라이머를 이용하여 증폭시킨 PCR산물은 약 950 bp의 cDNA가 얻어졌고, 이를 이용하여 조사한 RFLP패턴은 ER-PSV와 같았다. 이와 같은 RFLP분석과 Er-PSV의 항혈청을 이용한 혈청학적 성질의 결과로부터 Tr-PSV는 ER-PSV와 같은 서브그룹 I에 속하는 PSV의 한 계통으로 판단되었다. 국내에서 토끼풀로부터 PSV가 분리 동정된 것은 이 논문이 처음이다.