玉米细菌性枯萎病菌改良Dot-ELISA检测研究

Study on detection of Pantoea stwartii subsp. stewartii based on Dot-ELISA

摘要:

通过激光切割技术加工出硝酸纤维素膜小圆片并粘贴在已打孔的塑料胶条上, 制成包含8个圆片的NCM条(NCM test strip), 进一步在NCM条上建立了玉米细菌性枯萎病菌的Dot-ELISA检测方法。研究发现, 在NCM圆片上的Dot-ELISA检测灵敏度与点样量密切相关, 采用10 mL/点的加样量比通常的1 mL/点可提高检测灵敏度10-100倍；间接Dot-ELISA的检测灵敏度是双抗夹心dot-ELISA的10倍, 该结果进一步在微孔板ELISA的检测中得到证实。玉米细菌性枯萎病菌的改良Dot-ELISA检测是一种较为灵敏、快速、稳定、规范的实验方法, 为进一步研究该病菌的微流控芯片斑点免疫检测方法奠定了前期工作基础。

Abstract:

Enzyme linked immunosorbent assay performing on newly modified nitrocellulose membrane strip (NCM test strip) for detection of Pantoea stwartii subsp.stewartii, the casual agent of bacterial wilt of corn, was established. The test strip used in this study has eight round pieces of nitrocellulose membrane with a diameter of 2 mm, which was cut with a VersaLaser, and then was affixed on a plastic adhesive tape. Detection sensitivity for the pathogen was increased by 10-100 folds when 10μL per site of the bacterial suspension was used, as compared to that in the normal amount of 1uL per dot; detection sensitivity in indirect dot-ELISA was 10 folds higher than that in sandwich double antibodies dot-ELISA (DAS-dot- ELISA)，These results were further confirmed from the ELISA tests carried on the normal microtiter plates. This study will promote development of microfluidic chip detection method combined with the dot immunoassay for P. stwartii subsp.stewartii on corn.