摘要
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 antigencoated-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.
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 antigencoated-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.
基金
supported by Public Science and Technology Research Funds Projects of Agriculture (201303021)
the National Basic Research Program (973) of China (No.2014CB138400)
