• Research in Plant Disease
• /
• v.15 no.2
• /
• pp.57-62
• /
• 2009

Genetic diagnosis method of Virion Captured (VC)/RT-PCR for Rice stripe virus (RSV) and Rice black-streaked dwarf virus (RBSDV), Korean major rice viruses transmitted by small brown plant hopper, Laodelphax striatellus, was developed. Virion extraction buffer for rice plant was 0.01M potassium phosphate buffer, pH 7.0, containing 0.5% sodium sulfite. However, the extraction buffer for L. striatellus was 0.01M potassium phosphate buffer, pH 7.0, containing 0.5% sodium sulfite and 2% polyvinylpyrrolidone wt 40,000 (PVP-40). Specific primers for detection of RSV and RBSDV were selected for VC/RT-PCR method. The specific primers were used as a duplex primer to detect viruliferous small brown plant hopper collected from Gimpo, Pyeongtaek and Siheung areas in Gyeonggi province. The genetic diagnosis methods of single and duplex VC/RT-PCR for RSV and RBSDV could be used easily and economically, especially on the diagnosis of L. striatellus. The rate of viruliferous insect (RVI) for RSV was compared with ELISA and VC/RT-PCR for L. striatellus collected from fields. RVI by ELISA was same as 9.2% with RVI by VC/RT-PCR. However, there were some different detection results between the methods. It could be suggested that there is a possibility of serological and/or genomic differences among RSV isolates. The portion of RVI detected simultaneously by ELISA and VC/RT-PCR was 71.0%, and the detection rate from VC/RT-PCR was higher as 3.2% than that from ELISA, which had a reason of simultaneous detection ability both RSV and RBSDV of VC/RT-PCR.

• Food Science of Animal Resources
• /
• v.31 no.2
• /
• pp.158-165
• /
• 2011

A sensitive reverse transcriptase-PCR (RT-PCR) method to detect viable Escherichia coli O157:H7 in milk was established. The primer sets were designed based on the nucleotide sequences of the rfbE (per) and wbdN genes in the O157 antigen gene cluster of E. coli O157:H7. RT-PCR using five different primer sets yielded DNA with sizes of 655, 518, 450, and 149-bp, respectively. All five of the E. coli O157:H7 strains were detected by RT-PCR, but 11 other bacterial species were not. The sensitivity of RT-PCR was improved by adding yeast tRNA as a carrier to the crude RNA extract. The RT-PCR amplifying the 149-bp DNA fragment was the most sensitive for detecting E. coli O157:H7 and the most refractory to the bactericidal treatments. Heat treatment at $65^{\circ}C$ for 30 min was the least inhibitory of all bactericidal treatments. Treatment with RNase A strongly inhibited the RT-PCR of heated milk but not unheated milk. This study described RT-PCR methods that are specific and sensitive with a detection limit of 10 E. coli O157:H7 cells, and showed that pre-treating milk samples with RNase A improved the specificity to detect viable bacteria by RT-PCR.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.8
• /
• pp.1165-1169
• /
• 2012

In April 2009, the H1N1 pandemic influenza virus emerged as a novel influenza virus. The aim of this study was to compare the performances of several molecular assays, including conventional reverse transcription polymerase chain reaction (RT-PCR), two real-time reverse transcription (rRT)-PCRs, and two multiplex RTPCRs. A total of 381 clinical specimens were collected from patients (223 men and 158 women), and both the Seeplex RV7 assay and rRT-PCR were ordered on different specimens within one week after collection. The concordance rate for the two methods was 87% (332/381), and the discrepancy rate was 13% (49/381). The positive rates for the molecular assays studied included 93.1% for the multiplex Seeplex RV7 assay, 93.1% for conventional reverse transcription (cRT)-PCR, 89.7% for the multiplex Seeplex Flu ACE Subtyping assay, 82.8% for protocol B rRT-PCR, and 58.6% for protocol A rRT-PCR. Our results showed that the multiplex Seeplex assays and the cRT-PCR yielded higher detection rates than rRT-PCRs for detecting the influenza A (H1N1) virus. Although the multiplex Seeplex assays had the advantage of simultaneous detection of several viruses, they were time-consuming and troublesome. Our results show that, although rRT-PCR had the advantage, the detection rates of the molecular assays varied depending upon the source of the influenza A (H1N1)v virus. Our findings also suggest that rRT-PCR sometimes detected virus in extremely low abundance and thus required validation of analytical performance and clinical correlation.

• Korean Journal Plant Pathology
• /
• v.12 no.4
• /
• pp.432-436
• /
• 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
• /
• v.12 no.2
• /
• pp.139-143
• /
• 2006

Virion captured reverse transcription polymerase chain reaction (VC/RT-PCR) could detect plant virus quickly and accurately. In the VC/RT-PCR, no antibody is needed unlike immuno-captured RT-PCR (IC/RT-PCR) which had been improved method of RT-PCR for plant viruses, and virus nucleic acids can be obtained easily within 30minutes by property of polypropylene PCR tube which is hold and immobilized viral particles on its surface. For the virion capture of Tomato spotted wilt virus (TSWV), the extraction buffer was tested. The optimum macerating buffer for TSWV was 0.01M potassium phosphate buffer, pH 7.0, containing 0.5% sodium sulfite. The viral crude sap was incubated for 30 min at $4^{\circ}C$. The virions in the PCR tubes were washed two times with 0.01M PBS containing 0.05% Tween-20. The washed virions were treated at $95^{\circ}C$ immediately for 1 min containing RNase free water and chilled quickly in the ice. Disclosed virions' RNAs by heat treatment were used for RT-PCR. Dilution end point of $10^{-5}$ from plant's crude sap infected with TSWV showed relatively higher detection sensitivity for VC/RT-PCR. During multiple detection using two or more primers, interference was arisen by interactions between primer-primer and plant species. The result of multiplex RT-PCR was influenced by combinations of primers and the kind of plant, and the optimum extraction buffer for the multiplex detection by VC/RT-PCR should be developed.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.8
• /
• pp.1398-1403
• /
• 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.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.7
• /
• pp.1021-1028
• /
• 2012

In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid, sensitive, and inexpensive detection of nervous necrosis virus (NNV) in olive flounder, Paralichthys olivaceus, in Korea. A set of six specific primers was designed to target the RNA 2 gene encoding the coat protein of Korean NNV strains. The RT-LAMP reaction successfully detected NNV after 30 min at $65^{\circ}C$. When the sensitivities among RT-LAMP, RT-PCR, and nested RTPCR were compared, the RT-LAMP was shown to be able to detect the RNA template at $2.58{\times}10^{-2}\;TCID_{50}/ml$, whereas the RT-PCR and nested RT-PCR were only able to detect the RNA template at $2.58{\times}10^2\;TCID_{50}/ml$ and $2.58TCID_{50}/ml$, respectively. Thus, the sensitivity of the RT-LAMP assay was higher than those of the RT-PCR assays. In the specificity test of the RT-LAMP, 2 genotypes of NNVs (SJNNV and RGNNV) were positive; however, no other fish viruses were positive with the primers, indicating that the RT-LAMP assay is only specific to NNV. A total of 102 olive flounder were collected from hatcheries between 2009 and 2011. The occurrence of NNV in olive flounder was determined to be 53.9% (55/102) by the RT-LAMP. On the other hand, the prevalence based on the nested RT-PCR and RT-PCR results was 33.8% (34/102) and 20.6% (21/102), respectively. This result indicates that the RT-LAMP assay developed in this study is suitable for early field diagnosis of NNV with high sensitivity.

• The Korean Journal of Nuclear Medicine
• /
• v.38 no.6
• /
• pp.511-515
• /
• 2004

Purpose: The sodium-iodide symporter (NIS) expression is an important factor in determining the sensitivity of radioiodine therapy in well-differentiated thyroid cancers. Several previous studies for the expression of NIS in thyroid tissues show diverse results. To investigate whether there is difference between methods in determining the expression of NIS in thyroid tissues of patients with thyroid nodules, we measured the expression ot NIS using two different methods (RT-PCR and immunoshistochemical staining) and compared the results. Materials & Methods: We measured the expression of NIS by reverse transcriptase-polymerase chain reaction (RT-PCR) and also by immunohistochemical staining using anti-NIS antibody in thyroid cancers and other benign thyroid diseases. We compared the results of each method. We included 19 papillary carcinomas, 1 follicular carcinoma, 7 medullary carcinoma, 4 adenomas and 7 nodular hyperplasias. Results: By RT-PCR analysis, 10 of 19 papillary carcinomas expressed NIS, but 1 follicular cancer didn't express NIS. By immunohistochemical staining, 15 of 19 papaillary carcinomas express NIS, but 1 follicular lancer didn't express NIS. There was a significant correlation between the semiquautitative results of RT-PCR and immunohistochemical staining of NIS expression. (p<0.01) Conclusion: Our data demonstrated that the expression of NIS in thyroid cancers and other benign diseases investigated by RT-PCR and immunohistochemical staining correlated well each other. However, by immunohistochemical staining, more NIS expression was found.

• The Journal of Korean Society of Virology
• /
• v.27 no.1
• /
• pp.49-57
• /
• 1997

We developed a sensitive, nested reverse transcription-polymerase chain reaction (RT-PCR) to detect Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in animal tissues. Total RNA was extracted from blood, lung or kidney samples of experimentally-infected hamsters by using the guanidine isothiocyanate buffer-acid phenol-chloroform method. Genus-reactive outer primers were derived from the consensus region of the G1 gene sequences of several hantaviruses. Serotype-specific primers were selected within the region amplified by the outer primers. To examine the sensitivity and specificity of the test, we diluted known quantities of Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in human or hamster immune sera before performing the nested RT-PCR. We could detect as little as 1 pfu of virus, even in the presence of high-titer neutralizing antibodies, and the serotype-specific primers amplified only homologous serotype viruses. RT-PCR with these primers demonstrated virus in the blood of experimentally-infected hamsters as early as four days to as late as 30 days after infection. A comparison of a standard immunofluorescent antibody screening test (IFAT) to nested RT-PCR with RNA extracted from lung or kidney tissues of the hamsters, demonstrated that RT-PCR to be more sensitive for identifying viruses in these tissues.

• Korean Journal of Veterinary Service
• /
• v.32 no.2
• /
• pp.103-109
• /
• 2009

Influeza type A virus have been worldwide problematic in animals as well as in humans. In this study, the use of reverse-transcriptase polymerase chain reaction (RT-PCR) was described for detecting influenza virus type A. The primer of RT-PCR was designed from an nonstructural (NS) gene of Influenza A virus. By RT-PCR, a product with the size of 189 bp was detected only when influenza virus type A was used as template. No products could be detected with Influenza virus type B as well as other respiratory pathogens. The detection limit of the RT-PCR was up to $10^{0.3}TCID_{50}$ which is comparable to the sensitivity of cell culture method. The RT-PCR could detect the influenza A virus from nasal turbinates of the ferrets infected with influenza virus type A not type B.