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基于 FUM21 基因的 PCR-ELISA 法检测轮枝镰孢菌。

Detection of Fusarium verticillioides by PCR-ELISA based on FUM21 gene.

机构信息

Department of Pathological Sciences, Center of Biological Sciences, State University of Londrina (UEL), CEP 86057-970, Londrina, Paraná, Brazil.

Department of Biotechnology and Biochemistry, Center of Exact Sciences, State University of Londrina (UEL), CEP 86057-970, Londrina, Paraná, Brazil.

出版信息

Food Microbiol. 2018 Aug;73:160-167. doi: 10.1016/j.fm.2018.01.020. Epub 2018 Jan 19.

Abstract

Fusarium verticillioides is a primary corn pathogen and fumonisin producer which is associated with toxic effects in humans and animals. The traditional methods for detection of fungal contamination based on morphological characteristics are time-consuming and show low sensitivity and specificity. Therefore, the objective of this study was to develop a PCR-ELISA based on the FUM21 gene for F. verticillioides detection. The DNA of the F. verticillioides, Fusarium sp., Aspergillus sp. and Penicillium sp. isolates was analyzed by conventional PCR and PCR-ELISA to determine the specificity. The PCR-ELISA was specific to F. verticillioides isolates, showed a 2.5 pg detection limit and was 100-fold more sensitive than conventional PCR. In corn samples inoculated with F. verticillioides conidia, the detection limit of the PCR-ELISA was 1 × 10 conidia/g and was also 100-fold more sensitive than conventional PCR. Naturally contaminated corn samples were analyzed by PCR-ELISA based on the FUM21 gene and PCR-ELISA absorbance values correlated positively (p < 0.05) with Fusarium sp. counts (CFU/g). These results suggest that the PCR-ELISA developed in this study can be useful for F. verticillioides detection in corn samples.

摘要

串珠镰刀菌是一种主要的玉米病原体和伏马菌素的生产者,与人类和动物的毒性作用有关。基于形态特征的真菌污染传统检测方法耗时且灵敏度和特异性低。因此,本研究的目的是开发一种基于 FUM21 基因的 PCR-ELISA 方法用于检测串珠镰刀菌。通过常规 PCR 和 PCR-ELISA 分析了 F. verticillioides、Fusarium sp.、Aspergillus sp. 和 Penicillium sp. 分离株的 DNA,以确定特异性。PCR-ELISA 特异性针对串珠镰刀菌分离株,检测限为 2.5 pg,比常规 PCR 灵敏 100 倍。在接种了串珠镰刀菌孢子的玉米样品中,PCR-ELISA 的检测限为 1×10 个孢子/g,比常规 PCR 灵敏 100 倍。基于 FUM21 基因的 PCR-ELISA 分析了受自然污染的玉米样品,PCR-ELISA 吸光度值与 Fusarium sp. 计数(CFU/g)呈正相关(p<0.05)。这些结果表明,本研究中开发的 PCR-ELISA 可用于玉米样品中串珠镰刀菌的检测。

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