• 식물병연구
• /
• 제19권3호
• /
• pp.220-225
• /
• 2013

본 연구에서는 식물검역바이러스 4종(TBRV, ArMV, CLRV 및 GFLV)을 RT-PCR과 nested PCR 방법으로 진단 할 수 있는 방법을 개발하였다. 본 연구에서 개발한 방법은 모두 같은 PCR 조건으로 검사자에게 편리성과 신속성을 높여줄 뿐 아니라, 돌연변이-양성대조구의 사용으로 실험 오염여부를 확인할 수 있어 더욱 정확하다. 개발한 방법으로 최근 3년 Nepovirus속 4종의 바이러스를 검사한 결과, 27건을 검출하여 검역처분 하였다. 본 연구 결과들은 앞으로도 수출입 식물에서 해당 바이러스들을 신속, 정밀하게 진단할 수 있는 방법으로 활용할 수 있을 것으로 기대된다.

• 식물조직배양학회지
• /
• 제25권6호
• /
• pp.519-524
• /
• 1998

한국산 수박 녹반 모자이크 바이러스(CGMMV-WK)를 TR-PCR 기술에 의해서 간편하고 확실한 진단방법을 구명하고 아울러 CGMMV의 외피단백질 유전자를 클로닝 하였다. 바이러스의 추출은 Lee등 (1996)의 간이 조즙액추출법을 변형하여 정제된 핵산 추출액을 사용하여도 RT-PCR이 양호하였으며, 20 pmol의 primer, reverse transcriptase (30 unit), Rnasin (5unit)이 첨가된 PCR 반응액에서 one step reaction으로 RT-PCR이 가능하였다. CGMMV의 진단을 위한 RT-PCR 조건으로 42$^{\circ}C$에서 45분간 cDNA를 합성하고 있어서 95$^{\circ}C$ 에서 2분간 per-denaturation하고 96$^{\circ}C$ 에서 30초, 6$0^{\circ}C$ 에서 30초 그리고 72$^{\circ}C$에서 1분간으로 36 cycle을 반응을 수행함으로서 간편하고 확실하게 CGMMV를 진단할 수 있었다. 또한 추출된 CGMMV의 외피단백질의 염기서열분석 한 결과 CGMMV-W와는 98.77%, CGMMV-SH와는 99.38%의 상동성을 가지고 있었으며 아미노산 서열은 모두 100%의 상동성을 가지고 있었다.

• Asian Pacific Journal of Cancer Prevention
• /
• 제17권12호
• /
• pp.5229-5235
• /
• 2016

Prostate cancer (PCa) remains one of the most widespread and perplexing of all human malignancies. Assessment of gene expression is thought to have an important impact on cancer diagnosis, prognosis and therapeutic decisions. In this context, we explored combined expression of PCa related target genes AMACR and PCA3 in 126 formalin fixed paraffin embedded prostate tissues (FFPE) from Moroccan patients, using quantitative real time reverse transcription-PCR (RT-qPCR). This quantification required data normalization accomplished using stably expressed reference genes (RGs). A panel of twelve RG was assessed, data being analyzed using GenEx V6 based on geNorm, NormFinder and statistical methods. Accordingly, the hnRNP A1 gene was identified and selected as the most stably expressed RG for reliable and accurate gene expression quantification in prostate tissues. The ratios of both PCA3 and AMACR gene expression relative to that of the hnRNP A1 gene were calculated and the performance of each target gene for PCa diagnosis was evaluated using receiver-operating characteristics. PCA3 and AMACR mRNA quantification based on RT-qPCR may prove useful in PCa diagnosis. Of particular interesting, combining PCA3 and AMACR quantification improved PCa prediction by increasing sensitivity with retention of good specificity.

• Pediatric Infection and Vaccine
• /
• 제7권1호
• /
• pp.113-119
• /
• 2000

Purpose : Rotavirus is a most common etiologic agent of pediatric gastroenteritis. The standard method to diagnose rotavirus infection was the detection of viral particles in specimens through electron microscopy. But it was complex. Enzyme immunoassay and latex agglutinin are preferred because they are relatively handy, inexpensive and take a short time, in comparison with electron microscopy. However, several reports have shown that the use of ELISA to diagnose rotavirus infection in neonates can result in false positive reactions. The main purpose of this study is to compare ELISA and RT-PCR in the diagnosis of neonatal rotavirus infection. Methods : Data presented in this study were obtained form 123 newborn babies in the nursery of the Fatima Hospital, Masan, Korea, form Jury to December, 1997. We obtained two samples of stool from each of the newborn babies and then performed the Rotazyme test and the RT-PCR. In the Rotazyme test, the results were interpreted according to visual findings. The samples were used for the RT-PCR test after at stock $-30^{\circ}C$ to identify rotavirus group A. The result of the two tests were compared. Results : The informations are divided into 73 males and females. Out of the total informations 15 were transferred from other hospitals. Their average gestational age was $38.5{\pm}1.6$ weeks. The average birth weight was $3134.8{\pm}539gm$. In the Rotazyme test, 75 samples turned out to be positive. Out of them, 55 samples(75.3%) were positive and 18 samples(24.7%) were negative in the RT-PCR. On the other hand, in the Rotazyme test, 50 samples turned out be negative. Out of them, 27 samples(54%) were positive and 23 samples(46%) were negative in the RT-PCR. Conclusion : Rotavirus infection in uncommon in neonates. The diagnosis based on visual findings using Rotazyme test has a disadvantage in the sense that it can result in false positive reactions and false negative reactions in the diagnosis of neonatal rotavirus infection.

• 한국임상수의학회지
• /
• 제23권3호
• /
• pp.300-307
• /
• 2006

In the present study, methods of the reverse transcription-polymerase chain reaction(RT-PCR) were evaluated for the rapid detection and differentiation of transmissible gastroenteritis virus(TGEV), porcine epidemic diarrhea virus(PEDV) and rotavirus in piglets suffering from diarrhea. For the purposes, the PCR conditions were first confirmed for the amplification of VP7 gene of rotavirus and N gene of TGEV and PEDV using each specific primers and their annealing temperature. Multiplex RT-PCR methods were further determined to distinguish these viral infections and the results are as follows. For the specific amplification of these viral genes, the reliable PCR condition was determined as 30 cycles of reaction consisting each 1 min of denature at $94^{\circ}C$, annealing at $42^{\circ}C$ and polymerization at $72^{\circ}C$ with 1.0 mM $MgCl_2$. It was able to differentiate these viral infections in the intestines and feces of piglets suffering from diarrhea by duplex PCR for TGEV and PEDV and single PCR for rotavirus with a primer-annealing temperature of $42^{\circ}C$. When the multiplex RT-PCR were undertaken for the field samples, 17 cases of PEDV and 5 cases of rotavirus infections were differential diagnosed in a total of 92 samples of intestines and feces of the piglets with diarrhea.

• The Plant Pathology Journal
• /
• 제40권2호
• /
• pp.125-138
• /
• 2024

Citrus yellow vein clearing virus (CYVCV) is a member of the Alphaflexiviridae family that causes yellow vein clearing symptoms on citrus leaves. A total of 118 leaf samples from nine regions of six provinces in Korea were collected from various citrus species in 2020 and 2021. Viral diagnosis using next-generation sequencing and reverse transcription polymerase chain reaction (RT-PCR) identified four viruses: citrus tristeza virus, citrus leaf blotch virus, citrus vein enation virus, and CYVCV. A CYVCV incidence of 9.3% was observed in six host plants, including calamansi, kumquat, Persian lime, and Eureka lemon. Among the citrus infected by CYVCV, only three samples showed a single infection; the other showed a mixed infection with other viruses. Eureka lemon and Persian lime exhibited yellow vein clearing, leaf distortion, and water-soak symptom underside of the leaves, while the other hosts showed only yellowing symptoms on the leaves. The complete genome sequences were obtained from five CYVCV isolates. Comparison of the isolates reported from the different geographical regions and hosts revealed the high sequence identity (95.2% to 98.8%). Phylogenetic analysis indicated that all the five isolates from Korea were clustered into same clade but were not distinctly apart from isolates from China, Pakistan, India, and Türkiye. To develop an efficient diagnosis system for the four viruses, a simultaneous detection method was constructed using multiplex RT-PCR. Sensitivity evaluation, simplex RT-PCR, and stability testing were conducted to verify the multiplex RT-PCR system developed in this study. This information will be useful for developing effective disease management strategies for citrus growers in Korea.

• 생명과학회지
• /
• 제26권5호
• /
• pp.620-631
• /
• 2016

바이로이드는 매우 작은 RNA 분자로 구성되어 있으며, 외피 단백질이 없고 오로지 식물에만 감염되어 질병을 유발한다. 바이로이드 감염 질병을 예방하거나 진단하는 것은 상당히 어려운 일이며, 이는 병징이 초기에는 발견되지 않고 수확기에 접어들어서 발견되기 때문이다. 한편, 혈청학적인 방법은 식물 병원체를 검출하기 위해 주로 사용되었으나 바이로이드는 핵산인 RNA로만 구성되어 있기 때문에 이 방법으로 검출할 수가 없다. 때문에 바이로이드를 검출하기 위해 주로 사용되는 방법은 분자 생물학적인 방법으로, 초기에는 바이로이드의 분자적인 크기와 구조적 특징을 이용한 겔 전기 영동 방법이 주로 사용되었다. 그 후에는 역전사 반응과 중합효소 연쇄반응을 접목시킨 역전사 중합효소 연쇄반응(RT-PCR) 방법이 활용되었고, 그에 대한 효율적인 결과 확인을 위해 형광 물질을 도입한 실시간 역전사 중합효소 연쇄반응(Real-time RT-PCR)이 도입되었다. 그러나 그들은 온도를 변화시키기 위한 값비싼 기기와 전문적인 인력이 필요함으로 현장에서는 활용되기가 어렵다. 최근 개발된 고리 기반의 등온 증폭법(Loop-mediated isothermal amplification)의 경우, 온도의 변화가 필요 없어 비싼 온도 조절 기기가 필요하지 않다. 또한 매우 높은 증폭 효율을 지니며 반응 시간이 짧은 등의 여러 장점을 지니고 있기에 최근 현장 진단용 기술에 도입되고 있다. 이러한 배경으로, 이 총설에서는 바이로이드 유발 질병에 대하여 요약하고 그에 대한 검출 및 진단 방법에 대한 연구 동향에 대하여 기술하였다.

• The Plant Pathology Journal
• /
• 제31권3호
• /
• pp.219-225
• /
• 2015

The primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been used to detect viral RNA molecules because of its simplicity and high sensitivity for a number of viruses. RT-LAMP for the detection of Potato virus X (PVX) was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) to demonstrate its advantages over RT-PCR. RT-LAMP reactions were conducted with or without a set of loop primers since one out of six primers showed PVX specificity. Based on real-time monitoring, RT-LAMP detected PVX around 30 min, compared to 120 min for RT-PCR. By adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. RT-LAMP was conducted using simple inexpensive instruments and a regular incubator to evaluate whether RNA could be amplified at a constant temperature instead of using an expensive thermal cycler. This study shows the potential of RT-LAMP for the diagnosis of viral diseases and PVX epidemiology because of its simplicity and rapidness compared to RT-PCR.

• 농업생명과학연구
• /
• 제43권6호
• /
• pp.105-109
• /
• 2009

경산 묘목단지는 대단위 과수종묘를 생산하고 있어 경쟁력 강화를 위하여 무독묘 보증 생산이 요구되고 있다. 특히 생산규모가 큰 사과종묘에 대한 빠르고 정확한 바이러스 진단이 시급하다. 본 연구에서는 사과바이러스 진단을 위하여 다량의 시료를 동시 분쇄할 수 있는 bead beater를 이용하였으며 분쇄 bead는 저가의 산업용 glass bead (0.4 mm 직경)를 일회용으로 채택하였다. RNA추출을 위하여서는 guanidine thiocyanate 용액이 Trizol 용액보다 효과적인 것으로 나타났으며 silica membrane tube의 이용으로 RNA추출 간편성을 높일 수 있었다. 사과바이러스는 RT-PCR에 의하여 검증하였다.

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