等温扩增技术快速检测棕榈疫霉

doi:10.11707/j.1001-7488.20161020

摘要/Abstract

摘要： [目的] 棕榈疫霉是农林业生产上的一种危害严重的病原菌，建立棕榈疫霉的快速分子检测技术对于预测疫病发生情况、及时采取有效的防治方法控制疫病的传播和流行、减少经济损失具有重要意义。[方法] 采用环介导等温核酸扩增技术（loop-mediated isothermal amplification，LAMP），建立了以基因间隔区（intergenic spacer，IGS）为靶标的快速、简单、特异和灵敏的棕榈疫霉菌的检测方法。以IGS基因为靶序列，利用环介导等温核酸扩增技术，设计了多组LAMP引物，同时建立并优化了LAMP反应体系与条件，最终筛选出1组扩增反应结果较好的LAMP引物，采用该组引物进行特异性、灵敏度和实际应用的验证。在扩增前加入染料羟基萘酚蓝（HNB）作为反应指示剂，根据HNB的颜色变化判定反应结果。[结果] 整个检测过程在等温64℃条件下仅需80 min即可通过肉眼直接目测试验结果。在特异性试验中，通过HNB显色只有在棕榈疫霉菌株中能观察到天蓝色的阳性反应，而在其他疫霉、腐霉和真菌的供试菌株仍为紫色的阴性反应；在灵敏度试验中，PpIGS-LAMP技术检测灵敏度达到10 fg.μL^-1目标菌纯DNA；在实际应用中，PpIGS-LAMP技术能够快速检测出采自田间的木薯根部土壤中目标菌和人工接种发病木薯组织样品的目标菌。[结论] 本研究在国内外首次采用环介导等温扩增技术检测棕榈疫霉，建立了1种快速、高灵敏度、高特异性、可视化的棕榈疫霉的LAMP检测方法。建立的棕榈疫霉菌的LAMP检测方法在80 min内可完成待检样品的检测过程，显著缩短检测时间，降低基层部门的检测成本，为棕榈疫霉引起疫病的检疫和所致病害的快速诊断提供了新的检测技术。

关键词: 棕榈疫霉, 环介导等温扩增, PpIGS, 羟基萘酚蓝

Abstract: [Objective] The oomycetes pathogen Phytophthora palmivora is one of the most devastating pathogens in agricultural and forestry production. It is important to establish suitable molecular methods for detecting the pathogen with a rapid, cost-effective and efficient assay in predicting the occurrence of P. palmivora, controlling the spread of P. palmivora and reducing the economic losses. [Method] In this study, the loop-mediated isothermal amplification (LAMP) assay was used to establish a method for visual detection of P. palmivora with targeting the IGS gene. Multiple sets of LAMP primers were designed according to the IGS target using the LAMP technique. The LAMP reaction system and conditions were optimized to select a definitive set of primers. The set of primers were also verified by the specificity, sensitivity and application of the LAMP assay. A visualization indicator, namely, hydroxynaphthol blue (HNB) was brought into the PpIGS-LAMP assays prior to amplification. [Result] The result showed that the PpIGS-LAMP assay efficiently amplified the target element in 80 min at 64℃. All isolates of Phytophthora species, Pythium species, and fungi were used to determine the specificity of the LAMP assay. A positive colour (sky blue) was only observed in the presence of P. palmivora by addition of hydroxynaphthol blue prior to amplification, whereas none of other isolates showed a colour change. The detection limit of the PpIGS-specific LAMP assay for P. palmivora was 10 fg·μL^-1 of genomic DNA per reaction. Furthermore, the naturally P. palmivora-infected soil samples collected from diseased cassava root and inoculated cassava root were evaluated for detection P. palmivora. [Conclusion] In conclusion, this is the first report of the application of the LAMP assay technique for the rapid and specific detection of P. palmivora. These results suggest that this IGS-specific LAMP provides a rapid, high sensitivity, high specificity, visualization for detecting P. palmivora in plants and in production fields. The PpIGS-LAMP assay developed in this research efficiently amplified the target element in 80 min at 64℃, significantly shortened the testing time and reduced the cost of the government. Establishment of PpIGS-LAMP method provides a new alternative means for the rapid detection of P. palmivora.

Key words: Phytophthora palmivora, loop-mediated isothermal amplification (LAMP), PpIGS, hydroxynaphthol blue (HNB)

中图分类号:

S763.15

戴婷婷, 吴小芹. 等温扩增技术快速检测棕榈疫霉[J]. 林业科学, 2016, 52(10): 161-166.

Dai Tingting, Wu Xiaoqin. A Method for Rapidly Identifying Phytophthora palmivora Using the LAMP Technique[J]. Scientia Silvae Sinicae, 2016, 52(10): 161-166.

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