Wheat dwarf disease caused by wheat dwarf virus (WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for WDV, the coat protein (CP) gene of WDV was cloned and expressed in Escherichia coli. The purified recombinant CP protein was immunized to BALB/c mice, and four hybridoma cell lines (i.e. 18G10, 9G4, 23F4 and 22A10) secreting anti-WDV monoclonal antibodies (MAbs) were obtained through the hybridoma technique. Using the prepared MAbs, an antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and a dot-ELISA were established for detecting WDV in wheat samples. The most sensitive ACP-ELISA based on MAb 23F4 or 22A10 was able to detect WDV in 1:163,840 (w/v, g/mL) diluted WDV-infected wheat plant crude extracts. The dot-ELISA based on MAb 23F4 was the most sensitive and able to detect the virus in 1:5,120 (w/v, g/mL) diluted wheat plant crude extracts. A total of 128 wheat samples were collected from wheat growing regions in the Shaanxi and Qinghai provinces, China, and were screened for the presence of WDV using two developed serological assays. Results from the survey showed that approximately 62% of the samples were infected with WDV. PCR followed by DNA sequencing and sequence alignment validated the results from the two serological assays. Therefore, we consider that these two serological detection methods can be significantly useful for the control of WDV in China.
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We are grateful to Dr. Xinshun Ding (Samuel Roberts Noble Foundation, Ardmore, USA) for his valuable comments and manuscript edits. This work was supported by Public Science and Technology Research Funds Projects of Agriculture (201303021), and the National Basic Research Program (973) of China (No. 2014CB138400).
MHZ prepared the MAbs and carried out the immunoassays. RC performed the PCR detection of wheat samples. XPZ and JXW conceived of the study, participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflict of interest.
Animal and Human Rights Statement
The animal experiments were performed in accordance with the Principles of the Helsinki accords and approved by the Animal Experimentation Ethics Committee of Zhejiang University, Hangzhou, China.
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