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首页> 外文期刊>Acta Horticulturae >Detection of plum pox potyvirus (PPV) by DNA enzyme immunoassay
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Detection of plum pox potyvirus (PPV) by DNA enzyme immunoassay

机译：DNA酶免疫法检测李痘病毒杯状病毒（PPV）

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摘要

The DNA enzyme immunoassay (DEIA, Trademark of Sorin Biomedica, Italy) was used to detect PPV-specific dsDNA pieces amplified by PCR after immunocapturing virions with polyclonal rabbit IgG from PPV-infected tobacco and plum. For PCR-amplification,2 18-mer primers, located within the coat protein region were used which led to the synthesis of a 806 bp fragment. The PCR-product was detected by a 44-mer oligonucleotide complementary to viral RNA that resided inside the primer-defined boundaries. Thecapture oligonucleotide was 5'-biotinylated and could be linked to a microtitre plate coated with streptavidin. Hybrid molecules formed between the capture oligonucleotide and the PCR-products were finally detected by an dsDNA-specific monoclonal antibody and subsequent incubation with an anti-mouse rabbit IgG labelled with alkaline phosphatase. The detection of the PCR-products by the dsDNA-specific antibody was 6-times more sensitive than the detection by agarose gel electrophoresis. When the DEIA procedure was compared with DAS-ELISA, it revealed a higher sensitivity and exceeded the dilution end point of ELISA by 2 two-fold dilutions. This resulted in a higher test-fidelity especially when mixed samples were assayed.
机译：DNA酶免疫测定法（DEIA，意大利Sorin Biomedica的商标）用于检测PPV特异的dsDNA片段，该片段由PCR扩增，并用PPV感染的烟草和李子中的多克隆兔IgG免疫捕获病毒体。为了进行PCR扩增，使用了位于外壳蛋白区域内的2个18聚体引物，其导致了806bp片段的合成。通过与位于引物定义的边界内的病毒RNA互补的44-mer寡核苷酸检测PCR产物。捕获的寡核苷酸是5'-生物素化的，可以与涂有链霉亲和素的微量滴定板连接。最后，通过dsDNA特异性单克隆抗体检测捕获的寡核苷酸和PCR产物之间形成的杂合分子，然后与碱性磷酸酶标记的抗小鼠兔IgG孵育。 dsDNA特异性抗体对PCR产物的检测灵敏度比琼脂糖凝胶电泳检测高出6倍。当将DEIA程序与DAS-ELISA进行比较时，它显示出更高的灵敏度，并且通过2倍的两倍稀释液超出了ELISA的稀释终点。这导致更高的测试保真度，尤其是在分析混合样本时。

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