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

• Research in Plant Disease
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• v.16 no.2
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• pp.121-124
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• 2010

Immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) was applied to the detection of Cucumber green mottle mosaic virus (CGMMV), Kyuri green mottle mosaic virus (KGMMV), and Zucchini green mottle mosaic virus (ZGMMV) on Cucurbitaceae crops. These seed-borne tobamoviruses were accurately detected from the infected leaves and seeds by IC-RT-PCR. This method was estimated to be about 100 times more sensitive than ELISA, and also it allowed the direct confirmation of ELISA results by using the captured antigens from a completed ELISA microwell. This convenient and reliable method could be used routinely for large-scale field surveys or seed tests of Cucurbitaceae crops.

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

• Research in Plant Disease
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• v.12 no.2
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• pp.139-143
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• 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
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• v.22 no.8
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• pp.1165-1169
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• 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.

• Food Science and Biotechnology
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• v.17 no.3
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• pp.651-654
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• 2008

In order to enhance the efficacy of norovirus detection by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR, this study developed a norovirus mRNA concentration method using poly oligo dT-conjugated magnetic beads. An efficient norovirus detection protocol was performed on commercial ham using 2 viral elution buffers (glycine buffer and Tris beef extract buffer) and 2 concentration solutions [polyethylene glycol (PEG) and zirconium hydroxide]. The different approaches were verified by RT-PCR and nested PCR. This method was performed on ham in less than 8 hr by artificial inoculation of serial dilutions of the virus ranging from 1,000 to 1 RT-PCR unit/mL. The viral extraction and concentration method had 10-fold higher sensitivity using the combination of Tris beef extract buffer and PEG as compared to glycine buffer and zirconium hydroxide. This method proved that RT-PCR and nested PCR have the sensitive ability to detect norovirus in commercial ham, in that norovirus was successfully detected in artificially contaminated samples at a detection level as low as 1-10 RT-PCR unit/mL. Overall, such a detection limit suggests this protocol is both quick and efficient in terms of its potential use for detecting norovirus in meat products.

• 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.

• Korean Journal of Agricultural Science
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• v.42 no.2
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• pp.99-103
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• 2015

Potato virus T (PVT) is a plant pathogen in the family Betaflexiviridae, group IV single-stranded positive sense RNA viruses. The major host of PVT is potato, and it has been reported in Ullucus tuberosus, Oxalis tuberosa and Tropaeolum tuberosum. This study aimed at developing reverse transcription (RT)-polymerase chain reaction (PCR) and nested PCR techniques for specific detection of PVT. Finally, Two RT-PCR primer sets were developed and verified. The RT-PCR products were amplified to 734 (PVT RT-PCR primer set 6) and 828 bp (PVT RT-PCR primer set 29) long to detect PVT. The nested PCR primer sets [PVT-N70/C20 ($734{\rightarrow}315bp$) and PVT-N75/C30 ($828{\rightarrow}529bp$)] were developed which are high sensitivity and verification for detection of PVT. Furthermore, a modified-positive control plasmid is use to verify contamination of laboratory in PVT detection. This study supported the diagnose PVT in potato or PVT related hosts.

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

• Korean Journal of Veterinary Service
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• v.39 no.2
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• pp.107-116
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• 2016

In the study, we developed and evaluated a uracil N-glycosylase (UNG)-supplemented single-tube nested reverse transcription-polymerase chain reaction (UsnRT-PCR) assay that can carried out first-round RT-PCR and second-round nested PCR in a reaction tube without reaction tube opening and can simultaneously detect EU- and NA-PRRSV. The UsnRT-PCR confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PRRSV DNA from previous UsnRT-PCR. Primer specificities were evaluated with RNAs extracted from 8 viral strains and our results revealed that the primers had a high specificity for both genotypes of PRRSV. The sensitivity of the UsnRT-PCR was 0.1 $TCID_{50}$/0.1 mL for EU- or NA-PRRSV, respectively, which is comparable to that of previously reported real time RT-PCR (RRT-PCR). Clinical evaluation on 110 field samples (60 sera and 50 lung tissues) by the UsnRT-PCR and the RRT-PCR showed that detection rates of the UsnRT-PCR was 70% (77/110), and was relatively higher than that of the RRT-PCR (69.1%, 76/110). The percent positive or negative agreement of the UsnRT-PCR compared to RRT-PCR was 96.1% (73/76) or 90.9% (30/33), showing that the test results of both assays may be different for some clinical samples. Therefore, it is recommend that diagnostic laboratory workers use the two diagnostic assays for the correct diagnosis for the relevant samples in the swine disease diagnostic laboratories. In conclusion, the UsnRT-PCR assay can be applied for the rapid, and reliable diagnosis of PRRSV without concerns about preamplified DNA carryover contamination that can occurred in PCR process in the swine disease diagnostic laboratories.