Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (6): 1154-1162.doi: 10.3864/j.issn.0578-1752.2021.06.007

• PLANT PROTECTION • Previous Articles     Next Articles

Mapping of Epitopes and Establishment of Rapid DAS-ELISA for Potato Virus Y Coat Protein

YuXin LIANG1,2(),JianXiang WU3,XiaoYu LI2,ChunYu ZHANG2,JiChao HOU1,XuePing ZHOU3,4,YongZhi WANG2,4()   

  1. 1College of Plant Protection, Jilin Agricultural University, Changchun 130118
    2Institute of Plant Protection, Jilin Academy of Agricultural Sciences, Changchun 130033
    3Institute of Biotechnology, Zhejiang University, Hangzhou 310058
    4Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2020-06-11 Accepted:2020-08-14 Online:2021-03-16 Published:2021-03-25
  • Contact: YongZhi WANG;


【Objective】Potato virus Y (PVY) is one of the most serious viruses that affect the yield and quality of potato. At present, no effective virus treatment agent has been found, and application of virus-free seed potato is the main control measure to prevent PVY damage. The objective of this study is to establish a rapid detection method with high sensitivity and specificity, and to provide technical support for quality control of virus-free seed potato. 【Method】PVY coat protein (CP) was expressed as small polypeptides with overlapping parts, and the epitopes of PVY-CP were analyzed by Western blot. Matching experiments were performed on monoclonal antibodies that recognized different epitopes by conventional double antibody sandwich ELISA (DAS-ELISA), and a pair of monoclonal antibodies with the best detection effect was screened. At the same time, the captured and detected antibodies were confirmed. According to the standard of maximum P/N value, the working concentration of antibody was determined by square titration. The optimal co-incubation time of antibody and antigen was determined by control variable method. The sensitivity of rapid DAS-ELISA was tested with different concentrations of PVY-CP protein as antigen. Potato samples infected with different viruses were used as antigens to detect the specificity of rapid DAS-ELISA. Through rapid DAS-ELISA and RT-PCR detection of 50 samples of potato samples with suspected infection PVY collected in the field, the coincidence rate was tested. Finally, the established rapid DAS-ELISA was used to detect potato samples infected by different PVY strains. 【Result】A panel of monoclonal antibodies (9G6 and 3D3) that can be used for DAS-ELISA were screened using the expressed peptides of PVY CP, and a rapid DAS-ELISA was established based on this panel of MAbs. The ELISA plate coated with the capture antibody 9G6, and then co-incubated with detector antibody HRP labeled 3D3 (2 μg·mL-1) and samples for 5 min at 37℃ after blocking the plate. The detection limit was 0.5 ng·mL-1. The results of specific analysis showed that the established method was only positive for potato samples infected with PVY, and negative for potato virus S (PVS), potato virus M (PVM) and potato leaf-roll virus (PLRV). In comparison with RT-PCR, DAS-ELSIA had the coincidence rate of 96% through testing 50 potato simples, and the results of PVYN and PVYO potato samples were positive. 【Conclusion】 The established PVY detection method has high sensitivity and specificity, and can be completed within 30 min, which is convenient and fast, and provides key technical support for high-throughput detection of PVY and the production of virus-free seed potato.

Key words: potato virus Y (PVY), monoclonal antibody, antigen epitope, double antibody sandwich ELISA (DAS-ELISA)

Table 1

Primers for PVY-CP protein segmented expression"

片段 Segment 引物 Primer 引物序列Primer sequence (5′-3′)

Fig. 1

Schematic diagram of the segmented expression of PVY-CP protein"

Table 2

Analysis of the antigen epitopes identified by 3 strains of PVY MAb"

Segmented expression protein
单克隆抗体Monoclonal antibody
3D3 9G6 3E4
PVY-CP1-180 + + +
PVY-CP90-281 + + -
PVY-CP1-59 - - +
PVY-CP31-89 - - +
PVY-CP90-148 + + -
PVY-CP120-180 + - -

Fig. 2

Optimization of rapid DAS-ELISA detection conditions The effect of capture antibody incubating condition on the rapid DAS-ELISA; The effect of incubation condition of mixture of sample and the detection antibody on the rapid DAS-ELISA"

Table 3

Determination of working concentration of antibody"

Concentration of detection antibody (μg·mL-1)
捕获抗体浓度Concentration of capture antibody (μg·mL-1)
4 2 1 0.5
2 12.72±0.95* 7.56±0.93 5.10±0.53 4.05±0.13
1 7.44±0.77 4.42±0.07 2.82±0.12 2.46±0.01
0.5 5.17±0.36 3.31±0.04 2.34±0.19 2.03±0.07
0.25 2.87±0.25 2.14±0.01 1.81±0.18 1.59±0.15

Fig. 3

Sensitivity curve of rapid DAS-ELSIA Detection range of PVY potato leaf; Sensitivity curve of PVY protein"

Fig. 4

Specificity analysis of rapid DAS-ELISA"

Fig. 5

Detection of different strains of PVY by rapid DAS-ELISA"

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