Potato virus M(PVM) is one of the common and economically important potato viruses in potato-growing regions worldwide. To investigate and control this viral disease, efficient and specific detection techniques are ne...Potato virus M(PVM) is one of the common and economically important potato viruses in potato-growing regions worldwide. To investigate and control this viral disease, efficient and specific detection techniques are needed. In this study, PVM virions were purified from infected potato plants and used as the immunogen to produce hybridomas secreting PVM-specific monoclonal antibodies(MAbs). Four highly specific and sensitive murine MAbs, i.e., 1 E1, 2 A5, 8 A1 and 17 G8 were prepared through a conventional hybridoma technology. Using these four MAbs, we have developed an antigen-coated plate(ACP)-ELISA, a dot-ELISA and a Tissue print-ELISA for detecting PVM infection in potato plants and tubers. PVM could be detected in infected potato plant tissue crude extracts diluted at 1:10 240(w/v, g mL^(–1)) by the dot-ELISA or at 1:163 840(w/v, g mL^(–1)) by the ACP-ELISA. The Tissue print-ELISA is the quickest and easiest assay among the three established serological assays and is more suitable for onsite large-scale sample detection. Detection results of the field-collected samples showed that PVM is currently widespread in the Yunnan and the Heilongjiang provinces in China. The field sample test results of the developed serological assays were supported by the results from RT-PCR and DNA sequencing. We consider that the newly established ACP-ELISA, dot-ELISA and Tissue print-ELISA can benefit PVM detection in potato plant and tuber samples and field epidemiological studies of PVM. These assays can also facilitate the production of virus-free seed potatoes and breeding for PVM-resistant potato cultivars, leading to the successful prevention of this potato viral disease.展开更多
基金supported by the National Key Research and Development Project of China(2017YFD0201604)the National Natural Science Foundation of China(31571976)。
文摘Potato virus M(PVM) is one of the common and economically important potato viruses in potato-growing regions worldwide. To investigate and control this viral disease, efficient and specific detection techniques are needed. In this study, PVM virions were purified from infected potato plants and used as the immunogen to produce hybridomas secreting PVM-specific monoclonal antibodies(MAbs). Four highly specific and sensitive murine MAbs, i.e., 1 E1, 2 A5, 8 A1 and 17 G8 were prepared through a conventional hybridoma technology. Using these four MAbs, we have developed an antigen-coated plate(ACP)-ELISA, a dot-ELISA and a Tissue print-ELISA for detecting PVM infection in potato plants and tubers. PVM could be detected in infected potato plant tissue crude extracts diluted at 1:10 240(w/v, g mL^(–1)) by the dot-ELISA or at 1:163 840(w/v, g mL^(–1)) by the ACP-ELISA. The Tissue print-ELISA is the quickest and easiest assay among the three established serological assays and is more suitable for onsite large-scale sample detection. Detection results of the field-collected samples showed that PVM is currently widespread in the Yunnan and the Heilongjiang provinces in China. The field sample test results of the developed serological assays were supported by the results from RT-PCR and DNA sequencing. We consider that the newly established ACP-ELISA, dot-ELISA and Tissue print-ELISA can benefit PVM detection in potato plant and tuber samples and field epidemiological studies of PVM. These assays can also facilitate the production of virus-free seed potatoes and breeding for PVM-resistant potato cultivars, leading to the successful prevention of this potato viral disease.
