三种重要水果褐腐病菌快速检测试纸的研制

doi:10.13560/j.cnki.biotech.bull.1985.2018-1006

摘要/Abstract

摘要： 以水果上3种重要的褐腐病菌,即美澳型核果褐腐菌（Monilinia fructicola）、核果褐腐菌（M. laxa）和仁果褐腐菌（M. fructigena）为检测对象,研究开发适用于口岸水果检疫的快速检测试纸。以硝酸纤维素膜为固相载体,以病菌翻译延长因子基因（Translation elongation factor 1-alpha,EF-1α）为检测靶标,生物素标记的PCR扩增产物为检测标记物,采用DNA-DNA杂交方式对3种褐腐病菌进行试纸法快速检测。分别以褐腐病菌DNA和本研究构建的EF-1α重组质粒DNA为阳性标准品检验试纸的特异性和灵敏度。试验结果表明,本研究开发的检测试纸能够同时特异地检出以上3种褐腐病菌,整个检测流程（包括DNA提取和扩增过程）在2 h内即可完成,最低检测限达到10 fg/µL。其特异性强、灵敏度高、稳定性好,且检测结果可肉眼判定,无需依赖昂贵设备,便于向基层检疫实验室推广使用。

关键词: Monilinia fructicola, M. laxa, M. fructigena, 试纸, 快速检测

Abstract: A rapid detection dipstick for fruit quarantine in port was studied and developed using three important brown rot pathogens（Monilinia fructicola,Monilinia laxa and Monilinia fructigena）on fruits. Using nitrocellulose membrane as the solid phase carrier,the translation elongation factor 1-alpha（EF-1α）of above 3 Monilinia species as detecting targets,and PCR amplification products of biotin marker as detecting markers,DNA-DNA hybridization was applied in the rapid detection of 3 Monilinia species with the developed dipstick. The specificity and sensitivity of the Monilinia dipstick（abbr. as MON dipstick below）were evaluated by using DNA of brown rot strains and the constructed recombinant plasmids of EF-1α as positive standard samples,respectively. The results showed that the MON dipstick specifically detected 3 Monilinia species;the whole detection process（including DNA extraction and amplification process）was completed in 2 h,and the lowest detection limit reached 10 fg/µL. In sum,the dipstick developed in this study is of high specificity,sensitivity and stability,and the detection result is visible by human eyes,thus it does not rely on expensive equipment and can be expanded and used in local quarantine laboratory.

Key words: Monilinia fructicola, Monilinia laxa, Monilinia fructigena, dipstick, rapid detection

林惠娇, 杨华卫, 古恒森, 蒋湘, 张海磊, 刘昱辰, 周而勋. 三种重要水果褐腐病菌快速检测试纸的研制[J]. 生物技术通报, 2019, 35(6): 205-212.

LIN Hui-jiao, YANG Hua-wei, GU Heng-sen, JIANG Xiang, ZHANG Hai-lei, LIU Yu-chen, ZHOU Er-xun. Development of Dipstick for the Rapid Detection of Three Important Monilinia Species on Fruits[J]. Biotechnology Bulletin, 2019, 35(6): 205-212.

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