Archives of Virology

, Volume 164, Issue 3, pp 691–697 | Cite as

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

  • Yingjie Liu
  • Yingli Wang
  • Qin Wang
  • Yanhui Zhang
  • Wanxia Shen
  • Ruhui Li
  • Mengji Cao
  • Lei Chen
  • Xue Li
  • Changyong ZhouEmail author
  • Yan ZhouEmail author
Original Article


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.



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).

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Ethical approval

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

Supplementary material

705_2018_4123_MOESM1_ESM.docx (210 kb)
Supplementary material 1 (DOCX 210 kb)


  1. 1.
    Catara A, Azzaro A, Davino M, Polizzi G (1993) Yellow vein clearing of lemon in Pakistan. In: Moreno P, da Graca JV, Timmer LW (eds) Proceedings 12th conference of the international organization of citrus virologist. New Delhi, India, pp 364–367Google Scholar
  2. 2.
    Alshami AAA, Ahlawat YS, Pant RP (2003) A hitherto unreported yellow vein clearing disease of citrus in India and its viral etiology. Indian Phytopathol 56(4):422–427Google Scholar
  3. 3.
    Önelge N (2002) First report of yellow vein clearing of lemons in Turkey. J Turk Phytopathol 32:53–55Google Scholar
  4. 4.
    Hashmian BSM, Aghajanzadeh S (2017) Occurrence of Citrus yellow vein clearing virus in citrus species in Iran. J Plant Pathol 99(1):290Google Scholar
  5. 5.
    Chen HM, Li ZA, Wang XF, Zhou Y, Tang KZ, Zhou CY, Zhao XY, Yue JQ (2014) First report of Citrus yellow vein clearing virus on lemon in Yunnan, China. Plant Dis 98(12):1747Google Scholar
  6. 6.
    Zhou Y, Chen HM, Cao MJ, Wang XF, Jin X, Liu KH, Zhou CY (2017) Occurrence, distribution and molecular characterization of Citrus yellow vein clearing virus in China. Plant Dis 101(1):137–143Google Scholar
  7. 7.
    Loconsole G, Önelge N, Potere O, Giampetruzzi A, Bozan O, Satar S, De Stradis A, Savino V, Yokomi RK, Saponari M (2012) Identification and characterization of Citrus yellow vein clearing virus, a putative new member of the genus Mandarivirus. Phytopathology 102(12):1168–1175Google Scholar
  8. 8.
    Chen HM, Wang XF, Zhou Y, Zhou CY, Guo J, Li ZA (2016) Biological characterization and RT-PCR detection of a new disease of Eureka lemon. J Plant Prot 42(4):557–563Google Scholar
  9. 9.
    Zhou Y, Ma DD, Chen HM, Wang XF, He SG, Zhou CY (2016) A rapid and efficient purification of Citrus yellow vein clearing virus by sucrose cushion ultracentrifugation. J Plant Pathol 98(1):159–161Google Scholar
  10. 10.
    Zhang YH, Wang YL, Wang Q, Cao MJ, Zhou CY, Zhou Y (2018) Identification of Aphis spiraecola as a vector of Citrus yellow vein clearing virus. Eur J Plant Pathol 152(3):841–844Google Scholar
  11. 11.
    Zhang YH, Liu CH, Wang Q, Wang YL, Zhou CY, Zhou Y (2018) Identification of Dialeurodes citri as a vector of Citrus yellow vein clearing virus in China. Plant Dis. Google Scholar
  12. 12.
    Liu Z, Sunzhu YJ, Zhou XP, Hong J, Wu JX (2017) Monoclonal antibody-based serological detection of Citrus yellow vein clearing virus in citrus groves. J Integr Agric 16(4):884–891Google Scholar
  13. 13.
    Bin Y, Li ZA, Wu JX, Wang XF, Zhou Y, Li TS, Yang FY, Zhou CY, Song Z (2018) Development of an immunochromatographic strip test for rapid detection of Citrus yellow vein clearing virus. Arch Virol 163:349–357Google Scholar
  14. 14.
    Liu KH, Chen HM, Zhou Y, Li ZA (2015) Establishment of RT-LAMP assay for detection of Citrus yellow vein clearing virus. Acta Hortic Sin 42(5):997–1002Google Scholar
  15. 15.
    Zhou Y, Chen HM, Wang XF, Li ZA, Zhou CY (2016) Development and application of nested RT-PCR assay for detection of Citrus yellow vein clearing virus. J Plant Prot 43(2):255–259Google Scholar
  16. 16.
    Chen HM, Zhou Y, Wang XF, Zhou CY, Yang XY, Li ZA (2016) Detection of Citrus yellow vein clearing virus based on a real time RT-PCR approach. Acta Hortic Sin 43:168–174Google Scholar
  17. 17.
    Cave L, Brothier E, Abrouk D, Bouda PS, Hien E, Nazaret S (2016) Efficiency and sensitivity of the digital droplet PCR for the quantification of antibiotic resistance genes in soils and organic residues. Appl Microbiol Biotechnol 100:10597–10608Google Scholar
  18. 18.
    Yan Y, Jia XJ, Wang HH, Fu XF, Ji JM, He PY, Chen LX, Luo JY, Chen ZW (2016) Dynamic quantification of avian influenza H7N9(A) virus in a human infection during clinical treatment using droplet digital PCR. J Virol Methods 234:22–27Google Scholar
  19. 19.
    Lee E, Lee KJ, Park H, Chung JY, Lee MN, Chang MH, Yoo J, Lee H, Kong SY, Eom HS (2018) Clinical implications of quantitative JAK2 V617F analysis using droplet digital PCR in myeloproliferative neoplasms. Ann Lab Med 38(2):147–154Google Scholar
  20. 20.
    Rutsaert S, Bosman K, Trypsteen W, Nijhuis M, Vandekerckhove L (2018) Digital PCR as a tool to measure HIV persistence. Retrovirology 15:16Google Scholar
  21. 21.
    Xu XL, Peng C, Wang XF, Chen XY, Wang Q, Xu JF (2016) Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize. Transgenic Res 25(6):855–864Google Scholar
  22. 22.
    Palumbo JD, O’Keeffe TL, Fidelibus MW (2016) Characterization of Aspergillus section Nigri species populations in vineyard soil using droplet digital PCR. Lett Appl Microbiol 63(6):458–465Google Scholar
  23. 23.
    Zhong X, Liu XL, Lou BH, Zhou CY, Wang XF (2018) Development of a sensitive and reliable droplet digital PCR assay for the detection of ‘Candidatus Liberibacter asiaticus’. J Integr Agric 17(2):483–487Google Scholar
  24. 24.
    Zhao Y, Xia Q, Yin Y, Wang Z (2016) Comparison of droplet digital PCR and quantitative PCR assays for quantitative detection of Xanthomonas citri subsp. citri. PLoS One 11:e0159004Google Scholar
  25. 25.
    Bander BW, Zalom FG, Jayanth M, Sudarshana MR (2016) Phylogeny of Geminivirus coat protein sequences and digital PCR aid in identifying Spissistilus festinus as a vector of Grapevine red blotch-associated virus. Phytopathology 106(10):1223–1230Google Scholar
  26. 26.
    Mehle N, Dobnik D, Ravnikar M, Novak MP (2018) Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains. Anal Bioanal Chem 410(16):3815–3825Google Scholar
  27. 27.
    Fronhoffs S, Totzke G, Stier S, Werner N, Rothe M, Bruning T, Koch B, Sachinidis A, Vetter H, Ko Y (2002) A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction. Mol Cell Probes 16(2):99–110Google Scholar
  28. 28.
    Yang Q, Xi J, Chen XX, Hu SH, Chen N, Qiao SL, Wan SG, Bao DK (2017) The development of a sensitive droplet digital PCR for quantitative detection of porcine reproductive and respiratory syndrome virus. Int J Biol Macromol S104:1223–1228Google Scholar
  29. 29.
    Abachin E, Convers S, Falque S, Esson R, Mallet L, Nougarede N (2018) Comparison of reverse-transcriptase qPCR and droplet digital PCR for the quantification of dengue virus nucleic acid. Biologicals 52:49–54Google Scholar
  30. 30.
    Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, Vessella RL, Tewari M (2013) Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 10:1003–1005Google Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Yingjie Liu
    • 1
    • 2
  • Yingli Wang
    • 1
    • 2
  • Qin Wang
    • 1
    • 2
  • Yanhui Zhang
    • 1
    • 2
  • Wanxia Shen
    • 1
  • Ruhui Li
    • 3
  • Mengji Cao
    • 1
    • 2
  • Lei Chen
    • 4
  • Xue Li
    • 4
  • Changyong Zhou
    • 1
    • 2
    Email author
  • Yan Zhou
    • 1
    • 2
    Email author
  1. 1.National Citrus Engineering Research Center, Citrus Research InstituteSouthwest UniversityChongqingChina
  2. 2.Academy of Agricultural Sciences, Southwest UniversityChongqingChina
  3. 3.USDA-ARS, National Germplasm Resources LaboratoryBeltsvilleUSA
  4. 4.Xinping Yi-Dai Autonomous County Agricultural BureauYuxiChina

Personalised recommendations