• The Plant Pathology Journal
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• 제38권2호
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• pp.159-166
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• 2022

Barley yellow dwarf virus (BYDV) has been a major viral pathogen causing significant losses of cereal crops including oats worldwide. It spreads naturally through aphids, and a rapid, specific, and reliable diagnostic method is imperative for disease monitoring and management. Here, we established a rapid and reliable method for isothermal reverse transcription recombinase polymerase amplification (RT-RPA) combined with a lateral flow strips (LFS) assay for the detection of BYDV-infected oat samples based on the conserved sequences of the BYDV coat protein gene. Specific primers and a probe for RT-RPA reacted and optimally incubated at 42℃ for 10 min, and the end-labeled amplification products were visualized on LFS within 10 min. The RT-RPA-LFS assay showed no cross-reactivity with other major cereal viruses, including barley mild mosaic virus, barley yellow mosaic virus, and rice black streaked dwarf virus, indicating high specificity of the assay. The sensitivity of the RT-RPA-LFS assay was similar to that of reverse transcription polymerase chain reaction, and it was successfully validated to detect BYDV in oat samples from six different regions and in individual aphids. These results confirm the outstanding potential of the RT-RPA-LFS assay for rapid detection of BYDV.

• The Plant Pathology Journal
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• 제34권6호
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• pp.575-579
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• 2018

Apple stem grooving virus (ASGV) is considered to cause the most economically important viral disease in pears in Korea. The current PCR-based methods used to diagnose ASGV are time-consuming in terms of target detection. In this study, a novel assay for specific ASGV detection that is based on reverse transcription-recombinase polymerase amplification is described. This assay has been shown to be reproducible and able to detect as little as $4.7ng/{\mu}l$ of purified RNA obtained from an ASGV-infected plant. The major advantage of this assay is that the reaction for the target virus is completed in 1 min, and amplification only requires an incubation temperature of $42^{\circ}C$. This assay is a promising alternative method for pear breeding programs or virus-free certification laboratories.

• The Plant Pathology Journal
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• 제36권5호
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• pp.497-502
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• 2020

Barley yellow dwarf virus (BYDV) is an economically important plant pathogen that causes stunted growth, delayed heading, leaf yellowing, and purple leaf tip, thereby reducing the yields of cereal crops worldwide. In the present study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for the detection of BYDV in oat leaf samples. The RT-RPA assay involved incubation at an isothermal temperature (42℃) and could be performed rapidly in 5 min. In addition, no cross-reactivity was observed to occur with other cereal-infecting viruses, and the method was 100 times more sensitive than conventional reverse transcription polymerase chain reaction. Furthermore, the assay was validated for the detection of BYDV in both field-collected oat leaves and viruliferous aphids. Thus, the RT-RPA assay developed in the present study represents a simple, rapid, sensitive, and reliable method for detecting BYDV in oats.

• 식물병연구
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• 제27권2호
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• pp.79-83
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• 2021

The aim of the present study was to develop a sensitive and specific detection method for the rapid detection of apple scar skin viroid (ASSVd) in apple leaves. The resulting reverse transcription recombinase polymerase amplification (RT-RPA) assay can be completed in 10 min at 42℃, is 10 times more sensitive than conventional reverse transcription polymerase chain reaction, and can specifically amplify ASSVd without any cross-reactivity with other common apple viruses, including apple stem grooving virus, apple stem pitting virus, and apple chlorotic leaf spot virus. The reliability of the RT-RPA assay was assessed, and the findings suggested that it can be successfully utilized to detect ASSVd in field-collected samples. The RT-RPA assay developed in the present study provides a potentially valuable means for improving the detection of ASSVd in viroid-free certification programs, especially in resource-limited conditions.

• 한국재료학회지
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• 제34권2호
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• pp.63-71
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• 2024

Slipchip offers advantages such as high-throughout, low cost, and simple operation, and therefore, it is one of the technologies with the greatest potential for high-throughput, single-cell, and single-molecule analyses. Slipchip devices have achieved remarkable advances over the past decades, with its simplified molecular diagnostics gaining particular attention, especially during the COVID-19 pandemic and in various infectious diseases scenarios. Medical testing based on nucleic acid amplification in the Slipchip has become a promising alternative simple and rapid diagnostic tool in field situations. Herein, we present a comprehensive review of Slipchip device advances in molecular diagnostics, highlighting its use in digital recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and polymerase chain reaction (PCR). Slipchip technology allows users to conduct reliable droplet transfers with high-throughput potential for single-cell and molecule analyses. This review explores the device's versatility in miniaturized and rapid molecular diagnostics. A complete Slipchip device can be operated without special equipment or skilled handling, and provides high-throughput results in minimum settings. This review focuses on recent developments and Slipchip device challenges that need to be addressed for further advancements in microfluidics technology.

• The Plant Pathology Journal
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• 제39권5호
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• pp.522-527
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• 2023

The occurrence of postharvest kiwifruit rot has caused great economic losses in major kiwifruit-producing countries. Several pathogens are involved in kiwifruit rot, notably Botryosphaeria dothidea, and Diaporthe species. In this study, a recombinase polymerase amplification (RPA) assay was developed for the rapid and sensitive detection of the pathogens responsible for posing significant threats to the kiwifruit industries. The RPA primer pairs tested in this study were highly specific for detection of B. dothidea and D. eres. The detection limits of our RPA assays were approximately two picograms of fungal genomic DNA. The optimal conditions for the RPA assays were determined to be at a temperature of 39℃ maintained for a minimum duration of 5 min. We were able to detect the pathogens from kiwifruit samples inoculated with a very small number of conidia. The RPA assays enabled specific, sensitive, and rapid detection of B. dothidea and D. eres, the primary pathogens responsible for kiwifruit rots in South Korea.

• 식물병연구
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• 제26권4호
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• pp.195-201
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• 2020

Xanthomonas oryzae pv. oryzae는 벼 흰잎마름병을 일으키는 세균이며, 벼 흰잎마름병은 벼 주요 재배지에서 꾸준하게 발생하고 있다. 본 연구에서는 벼흰잎마름병균을 신속하고 간편하게 검출하기 위해 등온증폭법 중 하나인 recombinase polymerase amplification (RPA)를 적용하여 현장진단 등을 위한 유전자기반 진단법을 개발하였다. RPA법은 짧은 시간의 등온 조건에서 유전자 증폭이 가능하다는 장점이 있다. 본 연구에서 개발한 벼 흰잎마름병 RPA 진단법은 39℃에서 5분간만 반응하면 목표유전자의 증폭이 이루어지므로 기존 진단법보다 신속하고 간편하며 우리나라에 존재하는 K1-K3a의 4종 레이스에 모두 적용가능하며, DNA 추출 없이 식물체 잎의 즙액으로 증폭반응 수행이 가능하며 기존 Taq 기반 PCR보다 약 10배 검출 민감도가 높고 고가의 thermal cycler 없이도 항온수조, 발열블록 혹은 체온을 이용한 증폭반응 수행이 가능하다. 벼흰잎마름병은 벼의 농작물재해보험 대상재해 병충해 중의 하나이므로 과학적 진단결과가 요구되나, 일선 농촌지도 현장에서는 유전자기반 진단을 위한 장비, 기술 등의 부족으로 분자진단이 어려웠다. 본 연구에서 개발한 벼 흰잎마름병 RPA 진단을 활용한다 면 병징 의심부위 마쇄액을 주형으로, 손으로 5분 동안 반응시키는 것만으로도 신속하고 간편하게 벼 흰잎마름병의 목표유전자 증폭산물을 형성하므로 벼 흰잎마름병 진단의 최일선에서 활용할 수 있을 것이다.

• The Plant Pathology Journal
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• 제38권6호
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• pp.665-672
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• 2022

Cymbidium mosaic virus (CymMV) is one of economically important viruses that cause significant losses of orchids in the world. In the present study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay combined with a lateral flow immunostrip (LFI) assay was developed for the detection of CymMV in orchid plants. A pair of primers containing fluorescent probes at each terminus that amplifies highly specifically a part of the coat protein gene of CymMV was determined for RT-RPA assay. The RT-RPA assay involved incubation at an isothermal temperature (39℃) and could be performed rapidly within 30 min. In addition, no cross-reactivity was observed to occur with odontoglossum ringspot virus and cymbidium chlorotic mosaic virus. The RT-RPA with LFI assay (RT-RPA-LFI) for CymMV showed 100 times more sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR). Furthermore, the RT-PCR-LFI assay demonstrated the simplicity and the rapidity of CymMV detection since the assay did not require any equipment, by comparing results with those of conventional RT-PCR. On-site application of the RT-RPA-LFI assay was validated for the detection of CymMV in field-collected orchids, indicating a simple, rapid, sensitive, and reliable method for detecting CymMV in orchids.

• Parasites, Hosts and Diseases
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• 제59권2호
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• pp.167-171
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• 2021

Haemonchosis remains a significant problem in small ruminants. In this study, the assay of recombinase polymerase amplification (RPA) combined with the lateral flow strip (LFS-RPA) was established for the rapid detection of Haemonchus contortus in goat feces. The assay used primers and a probe targeting a specific sequence in the ITS-2 gene. We compared the performance of the LFS-RPA assay to a PCR assay. The LFS-RPA had a detection limit of 10 fg DNA, which was 10 times less compared to the lowest detection limit obtained by PCR. Out of 24 goat fecal samples, LFS-RPA assay detected H. contortus DNA with 95.8% sensitivity, compared to PCR, 79.1% sensitivity. LFS-RPA assay did not detect DNA from other related helminth species and demonstrated an adequate tolerance to inhibitors present in the goat feces. Taken together, our results suggest that LFS-RPA assay had a high diagnostic accuracy for the rapid detection of H. contortus and merits further evaluation.

• Journal of Microbiology and Biotechnology
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• 제32권1호
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• pp.91-98
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• 2022

Tetanus is a potentially fatal public health illness resulted from the neurotoxins generated by Clostridium tetani. C. tetani is not easily culturable and culturing the relevant bacteria from infected wounds has rarely been useful in diagnosis; PCR-based assays can only be conducted at highly sophisticated laboratories. Therefore, a real-time recombinase polymerase amplification assay (Exo-RPA) was constructed to identify the fragments of the neurotoxin gene of C. tetani. Primers and the exo probe targeting the conserved region were designed, and the resulting amplicons could be detected in less than 20 min, with a detection limit of 20 copies/reaction. The RPA assay displayed good selectivity, and there were no cross-reactions with other infectious bacteria common in penetrating wounds. Tests of target-spiked serum and pus extract revealed that RPA is robust to interfering factors and has great potential for further development for biological sample analysis. This method has been confirmed to be reliable for discriminating between toxic and nontoxic C. tetani strains. The RPA assay dramatically improves the diagnostic efficacy with simplified device architecture and is a promising alternative to real-time PCR for tetanus detection.