Skip to main content

Advertisement

SpringerLink
Log in

Development and application of a universal and simplified multiplex RT-PCR assay to detect five potato viruses

  • Viral and Viroid Diseases
  • Published:
Journal of General Plant Pathology Aims and scope Submit manuscript

Abstract

Potato virus X (PVX), Potato virus Y (PVY), Potato leafroll virus (PLRV), Potato virus M (PVM), and Potato virus A (PVA) commonly cause mixed infections and major economic losses in potato crops, so a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay has been greatly needed to detect all five viruses simultaneously. Previously published primers were evaluated, and five pairs of highly specific primers were selected for use. The quintuplex RT-PCR assay was optimized for primer concentration, annealing temperature, number of PCR cycles, and extension time. Finally, the quintuplex RT-PCR assay was successfully established. The amplified target fragments of 711 base pairs (bp) for PVX, 447 bp for PVY, 336 bp for PLRV, 520 bp for PVM and 273 bp for PVA, and the viral origins of the PCR products were confirmed by sequencing. This system can be easily customized for simplex, duplex, triplex, tetraplex, and quintuplex RT-PCRs by changing only the primer pairs while keeping the final reaction volume and the concentrations of other reagents constant to detect any combination of PVX, PVY, PLRV, PVM, and PVA viruses. In this way, the detection systems can be customized to the characteristics of viral infections specific to China’s potato-producing regions for excellent detection efficiency and meeting the needs of potato producers. The detection limit of this assay was up to 10−3 dilution in tetraplex and quintuplex RT-PCR, and even higher in simplex, duplex, and triplex RT-PCR. The efficiency of the quintuplex RT-PCR was compared with that of a DAS-enzyme-linked immunosorbent assay to detect the five viruses in 320 field potato samples, 195 in vitro plantlets and pooled potato samples. This new multiplex RT-PCR method is not only effective but also faster, cheaper, more sensitive, and more reliable than DAS-ELISA. It is especially suitable for the testing of in vitro plantlets and pooled samples of postharvest seed tubers.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Bai YJ, Wen JZ, Yang MX, Yu DC, Gao YL, Fan GQ, Wang WZ, Huang YH (2007) Comparison of incidence of major potato viruses in southwest and northeast potato-producing regions in China. J Northeast Agr Univ 38:733–736

    Google Scholar 

  • Bostan H, Peker PK (2009) The feasibility of tetraplex RT-PCR in the determination of PVS, PLRV, PVX and PVY from dormant potato tubers. Afr J Biotechnol 8:4043–4047

    CAS  Google Scholar 

  • Chen YT, Ning H, Zhang M (2010) Quick detection of multiple potato viruses by multiplex RT-PCR. Hunan Agr Sci 11:75–77

    Google Scholar 

  • Cheng Q, Zhu YF, Shen YF, Wang EH, Ma ZJ (2010) Study on detection of potato virus S by RT-PCR. Amino Acids Biotic Resour 32:8–11

    CAS  Google Scholar 

  • Chung BN, Yoon JY, Palukaitis P (2013) Engineered resistance in potato against potato leaf roll virus, potato virus A and potato virus Y. Virus Genes 47:86–92

    Article  CAS  PubMed  Google Scholar 

  • Clark MF, Adams AN (1977) Characteristics of the microplate method of enzyme linked immunosorbent assay for the detection of plant viruses. J Gen Virol 34:475–483

    Article  CAS  PubMed  Google Scholar 

  • Crosslin JM, Hamlin LL (2011) Standardized RT-PCR conditions for detection and identification of eleven viruses of potato and potato spindle tuber viroid. Am J Pot Res 88:333–338

    Article  Google Scholar 

  • Crosslin JM, Hamm PB, Shiel PJ, Hane DC, Brown CR, Berger PH (2005) Serological and molecular detection of tobacco veinal necrosis isolates of Potato virus Y (PVYN) from potatoes grown in the western United States. Am J Pot Res 82:263–269

    Article  CAS  Google Scholar 

  • Dai J, Cheng JL, Huang T, Zheng X, Wu YF (2012) A multiplex reverse transcription PCR assay for simultaneous detection of five tobacco viruses in tobacco plants. J Virol Methods 183:57–62

    Article  CAS  PubMed  Google Scholar 

  • Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE (2000) Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 13:559–570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Faccioli G (2001) Control of potato viruses using meristem culture and stem-cutting cultures, thermotherapy and chemotherapy. In: Loebenstein G, Berger PH, Brunt A, Lawson RH (eds) Virus and virus-like diseases of potatoes and production of seed-potatoes. Kluwer, Dordrecht, pp 365–390

    Chapter  Google Scholar 

  • Faccioli G, Marani F (1998) Virus elimination by meristem tip culture and tip micrografting. In: Hadidi A, Khetarpal RK, Koganzawa H (eds) Plant virus disease control. APS Press, St. Paul, pp 346–380

    Google Scholar 

  • Fan GQ, Bai YJ, Gao YL, Zhang W, Zhang S, Shen Y, Liu K, Yu J (2014) Investigation and analysis on potato viral disease in China. J Northeast Agr Univ 44:74–79

    Google Scholar 

  • Gambino G, Gribaudo I (2006) Simultaneous detection of nine grapevine viruses by multiplex reverse transcription-polymerase chain reaction with coamplification of a plant RNA as internal control. Phytopathology 96:1223–1229

    Article  CAS  PubMed  Google Scholar 

  • Gao YL, Zhang W, Bai YJ, Shen Y, Fan GQ, Geng HW, Liu WT (2011) Investigation and analysis of viral diseases in main potato production areas. Plant Protect 37:149–151

    Google Scholar 

  • Ge B, Li Q, Liu G, Lu M, Li S, Wang H (2013) Simultaneous detection and identification of four viruses infecting pepino by multiplex RT-PCR. Arch Virol 158:1181–1187

    Article  CAS  PubMed  Google Scholar 

  • Hao AY, Zhang JJ, Shen JP (2007) Types of potato viruses and their control. Inner Mongolia Agr Sci Tech 2:62–63

    Google Scholar 

  • Hu XX, Lei Y, He CZ, Liu MY, Li YL, Song Y (2012) Species and epidemiology of virus in main potato regions in Hunan Province. Chin Potato J 26:358–361

    Google Scholar 

  • Huang P, Yan Q, Din GY (2009) Occurrence and control of potato virus S in Guizhou. Guizhou Agr Sci 37:88–89

    CAS  Google Scholar 

  • Huttinga H (1996) Sensitivity of indexing procedures for viruses and viroids. In: Andrews JH, De Boer SH, Tommerup IC (eds) Pathogen indexing technologies. Advances in botany research, incorporating advances in plant pathology, vol. 23. Academic Press, New York, pp 59–71

  • Jansky SH, Jin LP, Xie KY, Xie CH, Spooner DM (2009) Potato production and breeding in China. Potato Res 52:57–65

    Article  Google Scholar 

  • Karasev AV, Nikolaeva OV, Hu X, Sielaff Z, Whitworth J, Lorenzen JH, Gray SM (2010) Serological properties of ordinary and necrotic isolates of potato virus Y: a case study of PVYN misidentification. Am J Pot Res 87:1–9

    Article  Google Scholar 

  • Liu WP (2007) Synergistic effect of potato virus Y (PVY) and potato spindle tuber viroid (PSTV) on tuber yield of potato. J Heilongjiang August First Land Reclam Uni 19:40–43

    CAS  Google Scholar 

  • Loebenstein G, Berger PH, Brunt AA, Lawson RH (2001) Virus and virus-like diseases of potatoes and production of seed-potatoes. Kluwer, Dordrecht

    Book  Google Scholar 

  • Ma Z, Michailides TJ (2007) Approaches for eliminating PCR inhibitors and designing PCR primers for the detection of phytopathogenic fungi. Crop Prot 26:145–161

    Article  CAS  Google Scholar 

  • Majumder S, Baranwal VK, Joshi S (2008) Simultaneous detection of Onion yellow dwarf virus and Shallot Latent virus in infected leaves and cloves of garlic by duplex RT-PCR. J Plant Pathol 90:371–374

    CAS  Google Scholar 

  • Nie XH, Singh RP (2000) Detection of multiple potato viruses using an oligo(dT) as a common cDNA primer in multiplex RT-PCR. J Virol Methods 86:179–185

    Article  CAS  PubMed  Google Scholar 

  • Nie XH, Singh RP (2001) A novel usage of random primers for multiplex RT-PCR detection of virus and viroid in aphids, leaves, and tubers. J Virol Methods 91:37–49

    Article  CAS  PubMed  Google Scholar 

  • Peiman M, Xie C (2006) Sensitive detection of potato viruses, PVX, PLRV and PVS, by RT-PCR in potato leaf and tuber. Autralas Plant Dis Notes 1:41–46

    Article  CAS  Google Scholar 

  • Raigond B, Sharma M, Chauhan Y, Jeevalatha A, Singh BP, Sharma S (2013) Optimization of RT-PCR of duplex RT-PCR for simultaneous detection of potato virus Y and S. Potato J 40:22–28

    Google Scholar 

  • Russo P, Miller L, Singh RP, Slack SA (1999) Comparison of PLRV and PVY detection in potato seed samples tested by Florida winter field inspection and RT-PCR. Am J Pot Res 76:313–316

    Article  CAS  Google Scholar 

  • Singh RP, Nie X (2003) Multiplex virus and viroid detection and strain separation via multiplex reverse transcription polymerase chain reaction. Can J Plant Pathol 25:127–134

    Article  CAS  Google Scholar 

  • Singh M, Singh RP (1996) Factors affecting detection of PVY in dormant tubers by reverse transcription polymerase chain reaction and nucleic acid spot hybridization. J Virol Methods 60:47–57

    Article  CAS  PubMed  Google Scholar 

  • Singh RP, Kurz J, Boiteau G, Bernard G (1995) Detection of potato leafroll virus in single aphids by reverse transcription polymerase chain reaction and its potential epidemiological application. J Virol Methods 55:133–143

    Article  CAS  PubMed  Google Scholar 

  • Singh RP, Kurz J, Boiteau G (1996) Detection of stylet-borne and circulative potato viruses in aphids by duplex reverse transcription polymerase chain reaction. J Virol Methods 59:189–196

    Article  CAS  PubMed  Google Scholar 

  • Singh RP, Nie X, Singh M (2000) Duplex RT-PCR: reagent concentrations at reverse transcription stage affect the PCR performance. J Virol Methods 86:121–129

    Article  CAS  PubMed  Google Scholar 

  • Singh RP, Nie X, Singh M, Coffin R, Duplessis P (2002) Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR. J Virol Methods 99:123–131

    Article  CAS  PubMed  Google Scholar 

  • Slack SA, Singh RP (1998) Control of viruses affecting potatoes through seed potato certification programs. In: Hadidi A, Khetarpal RK, Koganzawa H (eds) Plant virus disease control. APS, St. Paul, pp 249–260

    Google Scholar 

  • Sun Q, Zhang CQ, Meng ZD, Zhang FJ, Mu CH, Li WC, Zhang QW (2009) Establishment on the methods of detecting potato virus X and Y by complex RT-PCR. J Agr Biotech 17:737–738

    CAS  Google Scholar 

  • Wang XW (1999) Effect of infection status of potato virus Y and potato virus X on tuber yield of potato. Annual Reports, China’s Potato Association, pp 285–289

  • Wang Q, Zhang W (2010) An economic analysis of potato demand in China. Am J Pot Res 87:245–252

    Article  Google Scholar 

  • Wang XM, Jing LP, Yi H (2005) Advances in breeding of potato virus-resistant cultivars. Chin Potato J 19:285–289

    Google Scholar 

  • Wang B, MaYL Zhang ZB, Wu ZM, Wu YF, Wang QC, Li MF (2011) Potato viruses in China. Crop Prot 30:1117–1123

    Article  Google Scholar 

  • Wei T, Lu G, Clover G (2008) Novel approaches to mitigate primer interaction and eliminate inhibitors in multiplex PCR demonstrated using an assay for detection of three strawberry viruses. J Virol Methods 151:132–139

    Article  CAS  PubMed  Google Scholar 

  • Wei T, Lu G, Clover G (2009) A mutiplex RT-PCR for the detection of potato yellow vein virus, tobacco rattle virus and tomato infectious chlorosis virus in potato with a plant intemal amplification control. Plant Pathol 58:203–209

    Article  CAS  Google Scholar 

  • Xie KY, Qu DY, Jin LP, Pang WF (2008) Potato production in China and its comparison with the World. World Agr 349:35–41

    Google Scholar 

  • Xie Y, Wang M, Xu D, Li R, Zhoua G (2009) Simultaneous detection and identification of four sugarcane viruses by one-step RT-PCR. J Virol Method 162:64–68

    Article  CAS  Google Scholar 

  • Xu HM, Aubin JD, Nie JB (2010) Genomic variability in potato virus M and the development of RT-PCR and RFLP procedures for the detection of this virus in seed potatoes. Virol J 25:2–7

    Google Scholar 

  • Yan Q, Huang P, Song JX, Ding Y (2009) Preliminary investigation and identification of potato virus diseases at different altitudes in Guizhou. J Anhui Agr Sci 37:14263

    Google Scholar 

  • Yao BY, Wang GP, Ma XF, Liu WB, Tang HH, Zhu H, Hong N (2014) Simultaneous detection and differentiation of three viruses in pear plants by a multiplex RT-PCR. J Virol Methods 196:113–119

    Article  CAS  PubMed  Google Scholar 

  • Zhang W, Bai YJ, Gao YL, Shen Y, Fan GQ, Geng HW, Meng XX (2010a) A survey on occurrence frequencies of potato viruses in major potato-producing provinces in China. Heilongjiang Agr Sci 4:71–73

    Google Scholar 

  • Zhang W, Bai YJ, Shen Y, Gao YL, Fan GQ, Geng HW, Wang W, Meng XX (2010b) Cloning and sequences analysis of CP gene of potato virus X Heilongjiang isolate. Heilongjiang Agr Sci 8:1–5

    CAS  Google Scholar 

  • Zhou Y (2008) Detection and prevention of potato virus in Qinghai. Qinhai Sci Tech 3:14–16

    Google Scholar 

Download references

Acknowledgements

This research was supported by the China Agriculture Research System (CARS-10-P14) and the Harbin Technological Innovation Special Fund of China (2015RQQYJ031).

Author information

Authors and Affiliations

  1. Department of Plant Protection, College of Agriculture, Northeast Agricultural University, Harbin, 150030, People’s Republic of China

    Wei Zhang & Jingzhi Wen

  2. Virus-free Seedling Research Institute, Heilongjiang Academy of Agricultural Sciences, Harbin, 150086, People’s Republic of China

    Wei Zhang, Guoquan Fan, Yanling Gao, Yanju Bai, Cailing Qiu, Shu Zhang & Yu Shen

  3. State Key Laboratory of Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, People’s Republic of China

    Zhixiang Zhang

  4. Crop Breeding Institute, Heilongjiang Academy of Agricultural Sciences, Harbin, 150086, People’s Republic of China

    Xianxin Meng

Authors
  1. Wei Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhixiang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guoquan Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yanling Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jingzhi Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yanju Bai
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Cailing Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yu Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Xianxin Meng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jingzhi Wen or Yanju Bai.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, W., Zhang, Z., Fan, G. et al. Development and application of a universal and simplified multiplex RT-PCR assay to detect five potato viruses. J Gen Plant Pathol 83, 33–45 (2017). https://doi.org/10.1007/s10327-016-0688-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10327-016-0688-1

Keywords

Search

Navigation