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Reverse Transcription Droplet Digital PCR을 활용한 Tomato Spotted Wilt Virus 검출 및 정량
이효정(Hyo-Jeong Lee),박기범(Ki Beom Park),한연수(Yeon Soo Han),정래동(Rae-Dong Jeong) 한국식물병리학회 2021 식물병연구 Vol.27 No.3
식물 바이러스는 작물 수확량에 상당한 손실을 일으키고 작 물 생산을 지속적으로 위협하여 세계 식량 안보에 심각한 위협 이 된다. 그 중 tomato spotted wilt virus (TSWV)는 주로 원예 작물을 감염시키는 가장 위협적인 식물 바이러스로 넓은 기주 범위를 가진다. Reverse-transcription quantitative real-time PCR (RT-qPCR)은 TSWV의 민감한 검출을 위해 널리 사용되 고 있지만 표준화의 어려움으로 인해 유용성이 감소한다. 따라 서 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 특 이성 분석은 고추에서 주로 발생하는 바이러스 및 음성 대조군 에서 특이성을 확인한 결과 증폭되지 않았다. RT-ddPCR 및 RT- qPCR에 의해 측정된 TSWV의 선형회귀곡선은 모두 높은 선형 성을 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 특 이성을 제공하고 낮은 농도의 현장 시료에서 TSWV 검출하는 데 적합하다는 것을 보여준다. Plant viruses cause significant yield losses, continuously compromising crop production and thus represent- ing a serious threat to global food security. Tomato spotted wilt virus (TSWV) is the most harmful plant virus that mainly infects horticultural crops and has a wide host range. Reverse-transcription quantitative real-time PCR (RT-qPCR) has been widely used for detecting TSWV with high sensitivity, but its application is limited ow- ing to the lack of standardization. Therefore, in this study, a sensitive and accurate reverse transcription drop- let digital polymerase chain reaction (RT-ddPCR) method was established for TSWV detection. Additionally, we compared the sensitivities of RT-qPCR and RT-ddPCR for TSWV detection. Specificity analysis of RT-ddPCR for TSWV showed no amplification for main pepper viruses and negative control. TSWV transcripts levels measured by RT-ddPCR and RT-qPCR showed a high degree of linearity; however, the former yielded results that were at least 10-fold more sensitive and detected lower TSWV copy numbers than the latter. Collectively, our findings show that RT-ddPCR provides improved analytical sensitivity and specificity for TSWV detection, making it suitable for identifying low TSWV concentrations in field samples.
Metatranscriptomic Analysis of Plant Viruses in Imported Pear and Kiwifruit Pollen
이효정(Hyo-Jeong Lee),정래동(Rae-Dong Jeong) 한국식물병리학회 2022 Plant Pathology Journal Vol.38 No.3
Pollen is a vector for viral transmission. Pollenmediated viruses cause serious economic losses in the fruit industry. Despite the commercial importance of pollen-associated viruses, the diversity of such viruses is yet to be fully explored. In this study, we performed metatranscriptomic analyses using RNA sequencing to investigate the viral diversity in imported apple and kiwifruit pollen. We identified 665 virus-associated contigs, which corresponded to four different virus species. We identified one virus, the apple stem grooving virus, from pear pollen and three viruses, including citrus leaf blotch virus, cucumber mosaic virus, and lychnis mottle virus in kiwifruit pollen. The assembled viral genome sequences were analyzed to determine phylogenetic relationships. These findings will expand our knowledge of the virosphere in fruit pollen and lead to appropriate management of international pollen trade. However, the pathogenic mechanisms of pollen-associated viruses in fruit trees should be further investigated.
김나경(Na-Kyeong Kim),이효정(Hyo-Jeong Lee),김상민(Sang-Min Kim),정래동(Rae-Dong Jeong) 한국식물병리학회 2022 Plant Pathology Journal Vol.38 No.2
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 42oC 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.