| Potato is the world’s fourth major food crop after wheat, rice and corn. Potato viruses are the main pathogen of potato plants, and often harm potato in a co-infection way. Potato virus S (PVS), a member of the genus Carlavirus, and Potato virus Y (PVY), the type member of the genus Potyvirus, are two important potato viruses causing serious economic loss in potato industry. At present, planting virus-free seed potatoes is the major method to prevent and control potato virus diseases. However, sensitive and specific detection methods are the keys to breeding virus-free seed potatoes. Serological assays based on monoclonal antibody for plant virus detection are specific, rapid, sensitive, simple, and suitable to detect large-scale samples. So, it is currently the most important detection and diagnostic technique for plant viruses. In order to establish practical and accurate detection technique of PVS and PVY, monoclonal antibodies (MAbs) against PVS and PVY were respectively produced, and four serological assays based MAbs were respectively developed for rapid and sensitive detection of PVS and PVY in this study. Those study achievements will provide technology and material for virus detection and quarantine, breeding virus-free seed potatoes and establishment of scientific prevention and control systems of those two potato virus diseases.(1) MAbs against PVS and their detection application:purified PVS particles used as the immunogen, five hybridoma cell lines (1A3,16C10,18A9,20B12 and 22H4) secreting sensitive and specific MAbs against PVS were obtained by the hybridoma technique. The titers of ascitic fluids of MAbs secreted by the prepared hybridomas were up to 10-6 by indirect-ELISA. Western blot analyses indicated that all five MAbs could specifically react with 34 kDa coat protein of PVS. Based the prepared MAbs, four serological assays, antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA), double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and tissue blot enzyme-linked immunosorbent assay (Tissue blot-ELISA) were developed for detecting PVS in potato plants. Specificity analyses of the four developed serological assays demonstrated that all assays could accurately detect PVS in PVS-infected potato plant tissue extracts, but not the viruses in the extracts from PVY, PVA, Potato virus X (PVX), Potato leafroll virus (PLRV) infected plant tissues or healthy potato tissues. The sensitivity analyses revealed that the developed ACP-ELISA, DAS-ELISA and dot-ELISA could specifically detect virus in PVS-infected potato leaf tissue crude extracts diluted 1:81 920,1:163 840 and 1:10 240 (w/v, g/mL), respectively. Twenty-two potato field samples from Yunnan Province were detected for the presence of PVS by the developed serological assays. The detection results of serological assays indicated that 14 of the 22 potato field samples were infected by PVS. Further more, the serological detection result was consistent with the result of RT-PCR. PCR product sequencing and sequence comparative analysis proved that the positive samples detected by the serological assays were really infected by PVS.(2) MAbs against PVY and their detection application:purified PVY particles were used as the immunogen, and four hybridoma cell lines (3B2,3E4,20B2 and 25C2) secreting sensitive and specific MAbs against PVY were produced by the hybridoma technique. The titers of ascitic fluids of MAbs secreted by the prepared hybridomas were up to 10-6 by indirect-ELISA. Western blot analyses indicated that three MAbs (3E4、3B2、20B2) could specifically react with 30 kDa coat protein of PVY in PVY-infected plant tissues, while MAb 25C2 could react with an approximately 55 kDa protein supposed to be HC-Pro or VPg protein according to the molecular weight. Based the prepared MAbs, four serological assays, ACP-ELISA, dot-ELISA, Tissue blot-ELISA and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) were established to detect PVY in plants. Specificity analyses of the four developed serological assays demonstrated that all assays could accurately detect PVY in PVY-infected potato plant tissues, but not viruses in the extracts from PVS, PVA, PVX, PLRV-infected plant tissues or healthy potato and tobacco plant tissues. The sensitivity analyses indicated that the developed ACP-ELISA, dot-ELISA could specifically detect virus in PVY-infected plant tissue crude extracts diluted at 1:81 920 and 1:10 240 (w/v, g/mL), respectively. The developed IC-RT-PCR was the most sensitive, which could successfully detect virus in PVY-infected plant tissue crude extracts diluted 1:5 242 880 (w/v, g/mL). Thirty potato field samples from Yunnan Province were detected for the presence of PVYby the developed serological assays. The detection results of serological methods were same as the results of IC-RT-PCR and RT-PCR, and 20 of the 30 potato field samples were PVY-positive. PCR product sequencing and sequence comparative analysis proved that the positive samples detected by the serological assays were really infected by PVY... |
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