• 식물병연구
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• 제27권3호
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• pp.120-127
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• 2021

식물 바이러스는 작물 수확량에 상당한 손실을 일으키고 작물 생산을 지속적으로 위협하여 세계 식량 안보에 심각한 위협이 된다. 그 중 tomato spotted wilt virus (TSWV)는 주로 원예작물을 감염시키는 가장 위협적인 식물 바이러스로 넓은 기주 범위를 가진다. Reverse-transcription quantitative real-time PCR (RT-qPCR)은 TSWV의 민감한 검출을 위해 널리 사용되고 있지만 표준화의 어려움으로 인해 유용성이 감소한다. 따라서 본 연구에서는 TSWV 검출을 위해 민감하고 정확한 reverse transcription droplet digital polymerase chain reaction (RT-ddPCR)을 확립하였다. TSWV 검출에 대한 RT-qPCR 및 RT-ddPCR의 민감도를 비교하였고, TSWV에 대한 RT-ddPCR의 특이성 분석은 고추에서 주로 발생하는 바이러스 및 음성 대조군에서 특이성을 확인한 결과 증폭되지 않았다. RT-ddPCR 및 RTqPCR에 의해 측정된 TSWV의 선형회귀곡선은 모두 높은 선형성을 나타냈지만, RT-ddPCR 분석이 10배 이상 더 민감하고 더 낮은 TSWV의 copy 수를 검출할 수 있었다. 종합적으로, 우리의 연구 결과는 RT-ddPCR이 TSWV 검출에 대해 높은 민감도와 특이성을 제공하고 낮은 농도의 현장 시료에서 TSWV 검출하는 데 적합하다는 것을 보여준다.

• The Plant Pathology Journal
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• 제27권4호
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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.

• Journal of Microbiology and Biotechnology
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• 제28권2호
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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.

• Archives of Pharmacal Research
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• 제19권6호
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• pp.486-489
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• 1996

A simple, rapid, specific and sensitive method for the detection of serum hepatitis C virus (HCV) RNA using the reverse transcription-polymerase chain reaction (RT-PCR) technique without conventional RNA extraction was developed. HCV template RNA from serum was obtained by boiling the serum at $95^{\circ}C$ for 2 min, cooling rapidly in ice and removing the proteins by cetrifugation. RT-PCR amplifications including the reverse transcription and first PCR amplification were performed in one vessel containing both of reverse transcriptase and Taq DNA polymerase. The detection of HCV RNA from $10^{-3}{\mu}l$. serum was possible with this method. The suitability of this method for clinical analysis was evaluated by assaying HCV RNA in 225 patient samples including anti-HCV antibody negatives (13 samples) and positives (212 samples) by enzyme-linked immunosorbent assay test (ELISA). Detections of HCV RNA with this method were in 4 of 13 anti-HCV antibody negative samples (30.8%) and 95 of 212 positive samples (44.8%). The present method can be completed in 1 hr and has a wide range of application for the clinical utilities to determine the viral RNAS.

• 한국동물위생학회지
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• 제41권1호
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• pp.29-39
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• 2018

In this study, we developed a sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid visual detection of foot-and-mouth disease virus (FMDV) circulated in Korea. The RT-LAMP was completed in 40 min at $62^{\circ}C$ and the results of the assay were directly detected by naked eye without any detection process. The assay specifically amplified all 7 serotypes of FMDV RNAs but not amplified other viral and cellular nucleic acids. The sensitivity of the RT-LAMP was $10^2$, $10^3$ and $10^3TCID_{50}/mL$ for serotype O, A and Asia 1 FMDV, respectively, which was comparable to conventional reverse transcription polymerase chain reaction (RT-PCR) and relatively lower than that of real time quantitative RT-PCR (qRT-PCR). Clinical evaluation of the RT-LAMP using different serotypes of Korean and foreign FMDV strains showed a 100% (35/35) agreement with the results of the RT-PCR and qRT-PCR. These results indicated that RT-LAMP assay developed in this study could be a valuable diagnostic method for FMDV monitoring and surveillance.

• The Plant Pathology Journal
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• 제38권4호
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• pp.417-422
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• 2022

Apple stem grooving virus (ASGV) is a destructive viral pathogen of pome fruit trees that causes significant losses to fruit production worldwide. Obtaining ASGV-free propagation materials is essential to reduce economic losses, and accurate and sensitive detection methods to screen ASGV-free plantlets during in vitro propagation are urgently necessary. In this study, ASGV was sensitively and accurately quantified from in vitro propagated apple plantlets using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The optimized RT-ddPCR assay was specific to other apple viruses, and was at least 10-times more sensitive than RT-real-time quantitative PCR assay. Furthermore, the optimized RT-ddPCR assay was validated for the detection and quantification of ASGV using micropropagated apple plantlet samples. This RT-ddPCR assay can be utilized for the accurate quantitative detection of ASGV infection in ASGV-free certification programs, and can thus contribute to the production of ASGV-free apple trees.

• Journal of Plant Biotechnology
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• 제5권2호
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• pp.83-86
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• 2003

A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two specific 22-mer primers located in coat protein gene of SPFMV. A 411 bp PCR-product was detected in virus infected plants as well as tissue culture raised sweet potato but not in healthy plants. For optimization of RT-PCR protocol, the optimum crude nucleic acid concentration, annealing temperature, primer concentration and numbers of PCR-cycle for maximum sensitivity and specificity were determined. The optimum condition for RT-PCR was as follows: RT-PCR reaction mixture was one-step mixture, containing 50 pmol of primer, 30 units of reverse transcriptase, 5 units of RNasin, and the crude nucleic acid extracts (200 ng). In RT-PCR, cDNA was synthesized at $42^{\circ}C$ for 45 min before a quick incubation on ice after pre-denaturation at $95^{\circ}C$ for 5 min. The PCR reaction was carried out for 40 cycles at $96^{\circ}C$ for 30 see, $63^{\circ}C$ for 30 sec, $72^{\circ}C$ for 1 min, and finally at $72^{\circ}C$ for 10 min. The viral origin of the amplified product was confirmed by sequencing, with the sequence obtained having $95-98\%$ homology with published sequence data for SPFMV. The benefits of this RT-PCR based detection of SPFMV would be simple, rapid and specific.

• 한국미생물·생명공학회지
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• 제47권2호
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• pp.317-322
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• 2019

Gastroenteritis with diarrhea is one of the most infectious diseases in the world following respiratory infections. Notably, diarrhea-causing viruses (DVs) cause more than 70% of such cases. In this study, 3,065 stool specimens from patients with diarrhea (median age, 1.1 years; range, 0.0-91.1 years), who were admitted to the DanKook University Hospital, were examined using multiplex reverse transcription PCR (mRT-PCR). The target viruses were astrovirus (AstV), enteric adenovirus (EAdV), group A rotavirus (RotV), norovirus GI (NoV-GI), and norovirus GII (NoV-GII). The mRT-PCR results were analyzed based on various factors such as seasonality, age, presence of co-infection, and analyzed trends. The detection rate of the DVs during the study period was found to be 30.8% (n = 943/3,065). When the detection rate was analyzed monthly, the DV detection rate was found to be highest between December to January. Of the detected DVs, NoV-GII was the most common, accounting for 45.5% of the detected viruses (n = 446/980). Notably, 86.5% (n = 848/980) of the pathogens were detected in individuals who were less than 5 years of age. During the study period, NoV-GII and RotV showed alternating trends. In addition, both the number and rate of co-infections increased.

• Journal of Microbiology and Biotechnology
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• 제19권11호
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• pp.1470-1474
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• 2009

This paper describes the development a of direct multiplex reverse transcription-nested polymerase chain reaction (PCR) method, devised for simultaneous detection and typing of influenza viruses. This method combines the direct reverse transcription reaction without RNA purification with the enhancement of sensitivity and specificity of nested PCR. The method successfully detected three major human influenza viruses: influenza virus A subtype 1 (H1N1) and subtype 3 (H3N2), and influenza B virus (B). The minimum number of virus particles (pfu/ml) necessary for detection in spiked saliva samples was 200 (H1N1), 140 (H3N2), and 4.5 (B). The method's sensitivity and simplicity will be convenient for use in clinical laboratories for the detection and subtyping of influenza and possibly other RNA viruses.

• 대한수의학회지
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• 제57권1호
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• pp.37-42
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• 2017

Getah virus (GETV) infection causes sporadic outbreaks of mild febrile illness in horses and reproductive failure in pigs. In this study, we established a reverse transcription polymerase chain reaction (RT-PCR) method to detect GETV from suspected virus-infected samples. The reaction conditions were optimized and validated by using RNA extracted from GETV propagated in cell culture. A GETV-specific GED4 primer set was designed and used to amplify a 177 bp DNA fragment from a highly conserved region of the E1 glycoprotein gene in the GETV genome. RT-PCR performed with this primer set revealed high sensitivity and specificity. In the sensitivity test, the GED4 primer set detected GETV RNA at the level of $10^{2.0}\;TCID_{50}/mL$. In the specificity test, the GED4 primer set amplified only a single band of PCR product on the GETV RNA template, without non-specific amplification, and exhibited no cross-reactivity with other viral RNAs. These results suggest that this newly established RT-PCR method is useful for accurate identification of GETV infection in animals.