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

• Korean Journal of Veterinary Service
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• v.38 no.2
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• pp.107-116
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• 2015

In this study, we developed a rapid, sensitive and specific reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection of swine influenza viruse (SIV) including major subtypes of swine influenza viruses H1N1, H1N2 and H3N2, and a novel subtype of influenza A virus that accidentally infected in pig population. The RT-LAMP was completed in 40 min at $58^{\circ}C$ and the sensitivity of the RT-LAMP ($1copy/{\mu}L$) was 10-fold higher than conventional reverse transcription-polymerase chain reaction (RT-PCR) ($10copy/{\mu}L$) and the same to real time RT-PCR ($1copy/{\mu}L$). Also, the result of the RT-LAMP can be confirmed without any detection system. Therefore, the RT-LAMP could be a alternative diagnostic method for SIV detection in national SIV monitoring system and clinical diagnostic laboratory in the future.

• 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.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.198-204
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• 2002

Poliovirus is a member of enterovirus which causes paralytic poliomyelitis, encephalitis and aseptic meningitis. Since poliovirus is spread by the fecal-oral route and poliovirus-contaminated water could be a potential threat for public health, detection of poliovirus in drinking water resource is important. Infectious poliovirus and poliovirus inactivated by heat or UV were used to test three detection methods such as cell culture method, reverse transcription-polymerase chain reaction (RT-PCR) and integrated cell culture (ICC)-PCR. Infectious poliovirus was detected by all three methods and ICC-PCR was the most sensitive and fast in detecting poliovirus. Inactivated polioviruses could not be detected by cell culture or ICC-PCR methods. On the other hand, heat- inactivated viruses could be detected by RT-PCR. Thus it is suggested that ICC-PCR method is the most sensitive and effective in detecting infectious polioviruses in water sample.

• Journal of Microbiology and Biotechnology
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• v.30 no.3
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• pp.459-468
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• 2020

This study established a new polymerase spiral reaction (PSR) that combines with reverse transcription reactions for HCV detection targeting 5'UTR gene. To avoid cross-contamination of aerosols, an isothermal amplification tube (IAT), as a separate containment control, was used to judge the result. After optimizing the RT-PSR reaction system, its effectiveness and specificity were tested against 15 different virus strains which included 8 that were HCV positive and 7 as non-HCV controls. The results showed that the RT-PSR assay effectively detected all 8 HCV strains, and no false positives were found among the 7 non-HCV strains. The detection limit of our RT-PSR assay is comparable to the real-time RT-PCR, but is more sensitive than the RT-LAMP. The established RT-PSR assay was further evaluated for detection of HCV in clinical blood samples, and the resulting 80.25% detection rate demonstrated better or similar effectiveness compared to the RT-LAMP (79.63%) and real-time RT-PCR (80.25%). Overall, the results showed that the RT-PSR assay offers high specificity and sensitivity for HCV detection with great potential for screening HCV in clinical blood samples.

• Journal of Life Science
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• v.16 no.5
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• pp.729-733
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• 2006

Cryptosporidium is a waterborne pathogenic parasite which causes diarrhea. Immunomagnetic separation-immunofluorescent assay (IMS-IFA) has been a widely adopted for Cryptosporidium detection as standard method. However, this method does not provide information about viability or infectivity of Cryptosporidium. Therefore, many researchers have studied viability or infectivity analyses of Cryptosporidium with various methods such as vital staining, in vitro excystation, RT-PCR, cell culture, and mouse infection assay. In this study, two direct RT-PCR methods, cell culture RT-PCR and cell culture IFA were compared for sensitivity and other characteristics. The results showed that direct RT-PCR method with HSP70 genes had the highest sensitivity with detection up to 1 viable cell of Cryptosporidium. The infectious Cryptosporidium were detected up to 10 to 25 cells by cell culture methods in combination with RT-PCR and IFA. The infectious Cryptosporidium were apt to be quantified by cell culture IFA.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.143.2-143
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• 2003

A rapid and sensitive assay for the specific detection of plant viroids using reverse transcription-polymerase chain reaction(RT-PCR) has been developed already. The nested RT-PCR assay cloud be applied for the detection of apple scar skin viroid(ASSVd) from young leaves and other tissues. ASSVd has central conserved region(CCR), terminal left(T$\sub$L/) and terminal right(T$\sub$R/) domain. Primers were designed from these regions. Primer sets were successfully applicable for the amplification of full length or partial region of ASSVd by nested RT-PCR. Nested RT-PCR assay was more sensitive and accurate method to detect ASSVd from young trees during the early time of apple cultivation.

• Journal of Microbiology and Biotechnology
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• v.20 no.10
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• pp.1450-1456
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• 2010

Accurate and rapid diagnosis of Pandemic Influenza A/H1N1 2009 virus (H1N1 2009) infection is important for the prevention and control of influenza epidemics and the timely initiation of antiviral treatment. This study was conducted to evaluate the performance of several diagnostic tools for the detection of H1N1 2009. Flocked nasopharyngeal swabs were collected from 254 outpatients of suspected H1N1 2009 during October 2009. This study analyzed the performances of the RealTime Ready Inf A/H1N1 Detection Set (Roche), Influenza A (H1N1) Real-Time Detection Kit (Bionote), Seeplex Influenza A/B OneStep Typing Set [Seeplex Reverse Transcriptase PCR (RT-PCR)], BinaxNow Influenza A & B Test Kit [Binax Rapid Antigen Test (RAT)], and SD BIOLINE Influenza Ag kit (SD RAT). Roche and Bionote real-time RT-PCR showed identical results for the H1N1 2009 hemagglutinin gene. Compared with real-time RT-PCR, the sensitivities and specificities were 83.7% and 100% for Seeplex RT-PCR, 64.5% and 94.7% for Binax RAT, and 69.5% and 100% for SD RAT. The sensitivities of Seeplex RT-PCR, Binax RAT, and SD RAT in patients aged over 21 years were 73.7%, 47.4%, and 57.9%, respectively. The sensitivities of Seeplex RT-PCR, Binax RAT, and SD RAT on the day of initial symptoms were mostly lower (68.8%, 56.3%, and 31.3%, respectively). In conclusion, multiplex RT-PCR and RAT for the detection of H1N1 2009 were significantly less sensitive than real-time RT-PCR. Moreover, a negative RAT may require more sensitive confirmatory assays, because it cannot be ruled out from influenza infection.

• Journal of Practical Agriculture & Fisheries Research
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• v.5 no.1
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• pp.57-63
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• 2003

The bovine rotavirus(BRV), bovine coronavirus(BCV) and bovine viral diarrhea virus(BVDV) are main viruses of bovine viral diarrhea disease. These viruses could be rapidly amplified by the reverse transcriptase polymerase chain reaction(RT-PCR). This study was conducted to develop rapid and accurate diagnostic methods of these viral diseases by multiplex RT-PCR. Specific primers were designed based on the sequences reported by Chang KO et. al. (1997) and Schroeder BA, et. al. (1990), RNA were prepared from the cultured viruses, first-stranded DNAs were synthesised by reverse transcriptase. PCR were conducted to amplify specific regions of the viruses by multiplex. Three bands such as 1,062bp for BRV, 458bp for BCV, and 300bp for BVDV were successfully produced by multiplex RT-PCR. In conclusion, this result suggested that these viruses could be diagnosed rapidly and accurately by multiplex RT-PCR.

• The Plant Pathology Journal
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• v.31 no.3
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• pp.219-225
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• 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.