• Korean Journal Plant Pathology
• /
• v.14 no.4
• /
• pp.308-313
• /
• 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.

• Korean Journal Plant Pathology
• /
• v.12 no.3
• /
• pp.358-362
• /
• 1996

감자 잎말림 바이러스를 검정하기 위하여 ELISA 및 전자현미경에 의해 바이러스 감염이 확인된 기내 배양중인 감장의 줄기로부터 RT-PCR 분석을 수행하였다. 분리된 총 RNA들로부터 바이러스 cDNA를 합성하고 감자 잎말림 바이러스 외피단백질의 일부인 465bp를 특이하게 증폭하도록 고안한 두 primer를 사용하여 PCR 반응을 하였다. 증폭된 465pb의 DNA 절편의 염기서열을 분석한 결과 역시 감자 잎말림 바이러스임을 확인하였다. 바이러스 검정에 있어서 EL-ISA 방법과 RT-PCR 방법간의 민감도를 조사한 결과 RT-PCR 방법간의 민감도를 조사한 결과 RT-PCR 방법이 ELISA 방법보다 감자 잎말림 바이러스검정에 있어서보다 정확한 방법인 것으로 사료된다.

• Korean Journal Plant Pathology
• /
• v.14 no.3
• /
• pp.223-228
• /
• 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.

• Korean Journal of Veterinary Research
• /
• v.38 no.1
• /
• pp.85-89
• /
• 1998

본 연구에서는 돼지 로타바이러스의 혈청형 A, B 및 C를 동시에 진단할 수 있는 RT-PCR 기법을 개발하였다. 각각의 혈청형에 특이적인 primer를 이용하여 RT-PCR 기법으로 분변시료에서 직접 바이러스 동정을 실시한 결과, 23건의 로타바이러스 감염 분변 시료에서 13건이 혈청형 A, 3건이 혈청형 B, 2건이 혈청형 C로 나타나 국내에서도 A, B 및 C형의 돼지 로타바이러스가 공히 발생하고 있음을 확인하였으며, 발생분포는 외국의 예와 유사하였다. 이 RT-PCR기법은 돼지 로타바이러스의 주요 혈청형인 A, B 및 C형의 동시감별진단법으로 이용될 수 있을 것으로 판단된다.

• Korean Journal Plant Pathology
• /
• v.14 no.2
• /
• pp.130-135
• /
• 1998

This study was carried out to detect cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) in cultivated orchid plants in Korea. The standard double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) were carried out for detection of the viruses in the collected orchid samples. ELISA was suitable for massive-scale diagnostic method for virus detection in orchids. RT-PCR was rapid, time-saving and reliable detective method, and detection limit data showed that RT-PCR was 103 times more sensitive than ELISA. Of the 321 individual orchids representing 5 orchids genera tested by the ELISA, CymMV and ORSV were detected in 15.6% and 22.4%, and mixed infection of the both viruses with 4.9%, respectively. Of the Cymbidium plants tested, cultivated plants showed 52.5% virus infection rate with either CymMV or ORSV and both viruses.

• Korean Journal Plant Pathology
• /
• v.13 no.4
• /
• pp.219-224
• /
• 1997

A PT-PCR (reverse transcription-polymerase chain reaction) diagnostic method for potato virus Y (PVY) was developed using primer pair derived from conserved region of coat protein genes of several PVY strains, A 764 bp PCR product was detected from several lines of potato cv. Atlantic. We could prove that the 764 bp DNA fragment was indeed the PVY gene by sequencing analysis. PVY detection method using RT-PCR technique was about tuber tissue.

• Pediatric Infection and Vaccine
• /
• v.23 no.3
• /
• pp.194-201
• /
• 2016

Purpose: Monitoring pneumococcal carriage rates is important. We developed and evaluated the accuracy of a real-time reverse transcription polymerase chain reaction (RT-PCR) protocol for the detection of Streptococcus pneumoniae. Methods: In October 2014, 157 nasopharyngeal aspirates were collected from patients aged <18 years admitted to Chung-Ang University Hospital. We developed and evaluated a real-time PCR method for detecting S. pneumoniae by comparing culture findings with the results of the real-time PCR using genomic DNA (gDNA). Of 157 samples, 20 specimens were analyzed in order to compare the results of cultures, real-time PCR, and real-time RT-PCR. Results: The concordance rate between culture findings and the results of real-time PCR was 0.922 (P<0.01, Fisher exact test). The 133 culture-negative samples were confirmed to be negative for S. pneumoniae using real-time PCR. Of the remaining 24 culture-positive samples, 21 were identified as S. pneumonia -positive using real-time PCR. The results of real-time RT-PCR and real-time PCR from 20 specimens were consistent with culture findings for all S. pneumoniae -positive samples except one. Culture and real-time RT-PCR required 26.5 and 4.5 hours to perform, respectively. Conclusions: This study established a real-time RT-PCR method for the detection of pneumococcal carriage in the nasopharynx. Real-time RT-PCR is an accurate, convenient, and time-saving method; therefore, it may be useful for collecting epidemiologic data regarding pneumococcal carriage in children.

• Research in Plant Disease
• /
• v.24 no.3
• /
• pp.233-236
• /
• 2018

A multiplex RT-PCR (mRT-PCR) assay was developed for the detection of the recently reported viruses, Cherry virus A (CVA), Little cherry virus 1 (LChV-1), and Little cherry virus 2 (LChV-2), in cherry plants in Korea. Eight sets of primers were designed for each virus and their specificity was tested by using various combinations of mixed primer sets. From the designed primer sets, one combination was selected and further evaluated to estimate the optimum temperature and detection limits of the mRT-PCR. A newly developed mRT-PCR assay was also tested using 20 cherry samples collected in the field. This mRT-PCR assay may be a useful tool for field surveys of diseases and the rapid detection of these three viruses in cherry plants.

• Research in Plant Disease
• /
• v.23 no.2
• /
• pp.193-201
• /
• 2017

Major tomato viruses in Korea are Tomato chlorosis virus (ToCV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Pepper mottle virus (PepMoV), and Tomato mosaic virus (ToMV). RT-PCR conditions for the viruses were examined, especially in primer set and RT-PCR mixture. Total 46 primer sets from the unique sequence of the viruses were tested for nonspecific background products in a RT-PCR mixture without template. Among them 16 primer sets were applied to healthy tomato RNA, resulting the compatibility between RT-PCR mixture and primer set influenced RT-PCR to reduce nonspecific background products. Based on the combinations among cDNA synthesis parameters and RT-PCR mixtures, two reaction mixtures were finally selected for ToCV detection. The condition allowed to determine more specific primer sets; C029 (ToCV), C072 (TSWV), C070 (CMV), C048 (PepMoV), and C065 (ToMV). These primer sets are expected to be of use to specific detection of the major viruses in tomato plants.

• Asian Pacific Journal of Cancer Prevention
• /
• v.14 no.12
• /
• pp.7621-7628
• /
• 2013

Human epidermal growth factor receptor (HER) status is an important prognostic factor in breast cancer. There is no globally accepted method for determining its status, and which method is most precise is still a matter of debate. We here analyzed HER2 mRNA expression by quantitative reverse transcription-PCR (qRT-PCR) and HER2 DNA amplification using multiplex ligation-dependent probe amplification (MLPA). In parallel, we performed a routine evaluation of HER2 protein by immunohistochemistry (IHC). To assess the accuracy of the RT-PCR and MLPA techniques, a combination of IHC and fluorescence in situ hybridization (FISH) was used, substituting FISH when the results of IHC were ambiguous (2+) and for those IHC results that disagreed with MLPA and qRT-PCR, this approach being termed IHC-FISH. The IHC results for four samples were not compatible with the MLPA and qRT-PCR results; the MLPA and qRT-PCR results for these samples were confirmed by FISH. The correlations between IHC-FISH and qRT-PCR or MLPA were 0.945 and 0.973, respectively. The ASCO/CAP guideline IHC/FISH correlation with MLPA was (0.827) and with RT-PCR was (0.854). The correlations between the IHC results (0, 1+ as negative, and 3+ as positive) and qRT-PCR and MLPA techniques were 0.743 and 0.831, respectively. Given the shortcomings of IHC analysis and greater correlations between MLPA, qRT-PCR, and FISH methods than IHC analysis alone with each of these three methods, we propose that MLPA and real-time PCR are good alternatives to IHC. However a suitable cut-off point for qRTPCR is a prerequisite for determining the exact status of HER2.