• The Plant Pathology Journal
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• v.19 no.3
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• pp.159-161
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• 2003

The coat protein (CP) gene of Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was selected for the design of virus-specific primers for amplification and molecular detection of the virus in cultivated apple. A combined assay of reverse transcription and polymerase chain reaction (RT-PCR) was performed with a single pair of ApMV-specific primers and crude nucleic acid extracts from virus-infected apple for rapid detection of the virus. The PCR product was verified by restriction mapping analysis and by sequence determination. The lowest concentration of template viral RNA required for detection was 100 fg. This indicates that the RT-PCR for detection of the virus is a 10$^3$times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. The specificity of the primers was verified using other unrelated viral RNAs. No PCR product was observed when Cucumber mosaic virus (Cucumovirus) or a crude extract of healthy apple was used as a template in RT-PCR with the same primers. The PCR product (669 bp) of the CP gene of the virus was cloned into the plasmid vector and result-ant recombinant (pAPCP1) was selected for molecule of apple transformation to breed virus-resistant transgenic apple plants as the next step. This method can be useful for early stage screening of in vitro plantlet and genetic resources of resistant cultivar of apple plants.

• Journal of Microbiology and Biotechnology
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• v.27 no.4
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• pp.816-824
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• 2017

Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D's probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.210-217
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• 2018

A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.

• Journal of Korean Society of Environmental Engineers
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• v.29 no.8
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• pp.904-908
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• 2007

The protozoan pathogen Giardia lamblia has been major cause of waterborne enteric disease. In this study, we tried to identify G. lamlbia of human infectious species and to detect viable C. lamblia in river water samples including three sites of Han River mainstream and an its creek using PCR and RT-PCR technique. The PCR/RT-PCR methods were performed by using giardin primer based on the giardin gene targeting ventral disk of Giardia. Sensitivity testing in the DNA/RNA extraction and PCR/RT-PCR amplification steps showed that it was possible to detect a single cyst of G. lamblia and viable G. lamblia. The PCR/RT-PCR methods were compared with immunofluorescence(IF) assay by analyzing 48 samples collected from the mainstream water and the creek water. The mean concentration of the total cysts were 6.3 cysts/10 L(arithmetic mean, n = 48) and the positive detection rate were 62.5%(30/48). And the mean concentration of the cysts excluding empty cysts were 4.5 cysts/10 L and the positive detection rate were 52.1%(25/48). It resulted that 24 of 48 samples included Giardia lamblia by PCR assay and 10 of 48 samples included viable G. lamblia by RT-PCR assay. It resulted that the PCR/RT-PCR technique would be available to river water samples with low concentration of Giardia cysts. And it could support the Korean protozoan standard method, which provides useful information for species and viability.

• Biomedical Science Letters
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• v.25 no.4
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• pp.331-338
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• 2019

Human Aichivirus A1 (HuAiV-A1) is a waterborne human pathogenic virus classified as Picornaviridae and Kobuvirus. In this study, we developed a method that can detect about 35 minutes faster with the same detection sensitivity level than the previously reported HuAiV-A1 diagnostic RT-PCR primer. The RT-PCR primer sets developed in this study are capable of detecting HuAiV-A1 at a level of about 100 ag and formed 563 bp amplification product. In addition, the RT-nested PCR method was able to amplify 410 bp using the RT-PCR product as a template. The detection sensitivity of our method was 10 times higher than the method with the highest detection sensitivity to date. Therefore, the detection method of HuAiV-A1 developed in this study is expected to be used in the water environment in which a small amount of virus exists. Also, this detection method is expected to be used as HuAiV-A1 diagnostic technology in both clinical and non-clinical field.

• 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.

• Journal of Aquaculture
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• v.10 no.2
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• pp.171-178
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• 1997

Infectious pancreatic necrosis virus (IPNY) is an economically important fish pathogen since it causes the high-mortality disease in early stage of hatchery-reared fishes. In order to develop a rapid, sensitive and highly specific detection method for IPNV, reverse transcription-polymerase chain reaction (RT-PCR) was carried out using the oligonucleotide primers selected from the sequence of VP2, a major capsid polypertide of IPNV. As little as 40ng of purified IPNV dsRNA was detected by RT-PCR amplification, but no amplification products were obtained when nucleic acid genomes from other fish pathogens such as IHNV were used as RT-PCR templates. in situ RT-PCR methods are useful for the rapid and sensitive identification of IPNV.

• The Plant Pathology Journal
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• v.22 no.2
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• pp.164-167
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• 2006

An immunocapture reverse transcription-polymerase chain reaction (IC/RT-PCR) was developed for simultaneous detection of two pepper-infecting RNA viruses, Pepper mud mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV). The assay could be performed in a single tube for simultaneous and sensitive detection of these tobamoviruses. This detection system revealed thousand-fold increase in detection sensitivity compare to ELISA. This method could save time and reagent cost compare to common RT-PCR which needs several reactions and several procedures of viral RNA extractions for the same number of samples.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1110-1114
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• 2005

Six viruses of the genera Carlavirus (Garlic mosaic virus, GarMV, and Garlic latent virus, GarLV), Allexivirus (Garlic virus X, GarV-X, and Garlic mite-borne filamentous virus, GarMbFV) and Potyvirus (Leek yellow stripe virus, LYSV, and Onion yellow dwarf virus, OYDV) from Korean garlic plants with mosaic symptoms were simultaneously detected by multiplex RT-PCR and subsequently sequenced. An immunocapture RT-PCR for the detection of GarLV, LYSV, and OYDV was also performed. The coat protein phylogenetic analysis of the garlic viruses showed that the Korean isolates were most closely related to the isolates from China, Japan, Brazil, and Argentina. This study is the first report for the differentiation of six garlic viruses in Korea by simultaneous detection using multiplex RT-PCR.

• The Plant Pathology Journal
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• v.27 no.4
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• pp.385-389
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• 2011

A new reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the Potato leafroll virus (PLRV) was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) to address its advantages over RTPCR. RT-LAMP primers were designed from the open reading frame 3 (ORF3) sequence of PLRV. The RT-LAMP reactions were conducted without or with a set of loop primers. By real-time monitoring using Turbimeter, the RT-LAMP (with loop primers) detects PLRV in less than 30 min, compared to 120 min of RT-PCR. By adding fluorescent reagent during the reaction, final products of the RT-LAMP were fluorescently visualized under UV light or could be differentiated by naked-eye inspection under normal light. The RT-LAMP was extremely sensitive, about 2000-fold more sensitive than RT-PCR. This study presents great potential of the RT-LAMP for diagnosis and PLRV epidemiology because RT-LAMP method is speedy, sensitive, inexpensive, and convenient.