Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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Application of Reverse Transcription Droplet Digital PCR for Detection and Quantification of Tomato Spotted Wilt Virus

Reverse Transcription Droplet Digital PCR을 활용한 Tomato Spotted Wilt Virus 검출 및 정량

  • Lee, Hyo-Jeong (Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University) ;
  • Park, Ki Beom (Invirustech) ;
  • Han, Yeon Soo (Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University) ;
  • Jeong, Rae-Dong (Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University)
  • 이효정 (전남대학교 응용생물학과) ;
  • 박기범 ((주)인바이러스테크) ;
  • 한연수 (전남대학교 응용생물학과) ;
  • 정래동 (전남대학교 응용생물학과)
  • Received : 2021.08.23
  • Accepted : 2021.09.29
  • Published : 2021.09.30
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Abstract

Plant viruses cause significant yield losses, continuously compromising crop production and thus representing 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 owing to the lack of standardization. Therefore, in this study, a sensitive and accurate reverse transcription droplet 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.

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.

Keywords

Acknowledgement

This work was carried out with the support of Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ014947032021) Rural Development Administration, Republic of Korea.

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