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Development of a sensitive and reliable reverse transcription droplet digital PCR assay for the detection of citrus yellow vein clearing virus

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Abstract

In 2009, a new viral disease of citrus caused by citrus yellow vein clearing virus (CYVCV) was first discovered in China. CYVCV is considered to be the most serious pathogen affecting lemon production. In this study, a sensitive and reliable reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay was developed to detect and quantify CYVCV without references. The specificity of the assay was demonstrated by its failure to amplify other relevant citrus viruses. The quantitative linearity, sensitivity and accuracy of RT-ddPCR for detecting CYVCV were compared to those of real-time RT-PCR. The results showed that both methods had a high degree of linearity (R2 = 0.9776) and quantitative correlation. Furthermore, RT-ddPCR was found to be 100 times more sensitive than real-time RT-PCR, and it can therefore be used to detect CYVCV in individual arthropods. In summary, the results demonstrated that the RT-ddPCR assay is a promising approach for quantitative detection of CYVCV with high precision and accuracy.

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Funding

This work was supported in part by the Intergovernmental International Science, Technology and Innovation (STI) Collaboration Key Project of China’s National Key R&D Programme (NKP), The People’s Republic of China ministry of science and technology(CN) (2017YFE0110900), Overseas Expertise Introduction Project for Discipline Innovation (111 Center), Ministry of Education of the People’s Republic of China(CN) (B18044), Chongqing Research Program of Basic Research and Frontier Technology (cstc2015jcyjBX0043, cstc2017jcyjAX0150) and Fundamental Research Funds for the Central Universities, Ministry of Education of the People’s Republic of China (CN) (XDJK2018AA002).

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Authors and Affiliations

  1. National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing, 400712, China

    Yingjie Liu, Yingli Wang, Qin Wang, Yanhui Zhang, Wanxia Shen, Mengji Cao, Changyong Zhou & Yan Zhou

  2. Academy of Agricultural Sciences, Southwest University, Chongqing, 400712, China

    Yingjie Liu, Yingli Wang, Qin Wang, Yanhui Zhang, Mengji Cao, Changyong Zhou & Yan Zhou

  3. USDA-ARS, National Germplasm Resources Laboratory, Beltsville, MD, 20705, USA

    Ruhui Li

  4. Xinping Yi-Dai Autonomous County Agricultural Bureau, Yuxi, 653400, Yunnan, China

    Lei Chen & Xue Li

Authors
  1. Yingjie Liu
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  2. Yingli Wang
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  3. Qin Wang
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  4. Yanhui Zhang
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  5. Wanxia Shen
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  6. Ruhui Li
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  7. Mengji Cao
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  8. Lei Chen
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  9. Xue Li
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  10. Changyong Zhou
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  11. Yan Zhou
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Corresponding authors

Correspondence to Changyong Zhou or Yan Zhou.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Handling Editor: Jesús Navas-Castillo.

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Liu, Y., Wang, Y., Wang, Q. et al. Development of a sensitive and reliable reverse transcription droplet digital PCR assay for the detection of citrus yellow vein clearing virus. Arch Virol 164, 691–697 (2019). https://doi.org/10.1007/s00705-018-04123-7

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  • DOI: https://doi.org/10.1007/s00705-018-04123-7

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