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烟草黑胫病菌RPA检测方法的建立
杨懿德,范志朋,周婷,许大凤,李林秋,鄢敏,杨洋,羊国根,周本国,潘月敏
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(四川省烟草公司宜宾市公司,宜宾 644600;安徽农业大学植物保护学院,植物病虫害生物学与绿色防控安徽普通高校重点实验室,合肥 230036;安徽省农业科学院烟草研究所,合肥 230031)
摘要:
烟草黑胫病是由烟草疫霉(Phytophthora nicotianae)引起的烟草重要土传病害。建立了烟草黑胫病菌RPA快速检测方法,结合侧流层析试纸条,该方法在38 ℃恒温条件下30 min内完成可视化检测,不需要PCR仪等仪器设备。以烟草黑胫病菌YPT1为靶标基因设计RPA引物YPT1F和YPT1R,以及RPA探针YPT1-LFD-probe,对烟草黑胫病菌具有较高的检测特异性,其检测下限为10 pg·μL-1,与普通PCR一致。因此,建立的烟草黑胫病菌RPA检测技术具有快速、简单、实用等特点,为烟草黑胫病菌的快速检测提供了新的方法。
关键词:  烟草黑胫病菌  重组酶聚合酶技术  特异性  灵敏性
DOI:10.13610/j.cnki.1672-352x.20220504.001
基金项目:四川省烟草公司科技项目“烟草黑胫和根黑腐病原菌快检方法研究与产品开发”(SCYC201904)资助。
Development of recombinase polymerase amplification for detection of Phytophthora nicotianae
YANG Yide,FAN Zhipeng,ZHOU Ting,XU Dafeng,LI Linqiu,YAN Min,YANG Yang,YANG Guogen,ZHOU Benguo,PAN Yuemin
(Sichuan Tobacco Company Yibin City Company, Yibin 644600;School of Plant Protection, Key Laboratory of Biology and Sustainable Management of Plant Disease and Pests of Anhui Higher Education Institute, Anhui Agricultural University, Hefei 230036;Tobacco Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031)
Abstract:
Phytophthora nicotianae is a soil-borne phytopathogenic oomycete that causes tobacco black shank. In this study, the recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) technology for the rapid and sensitivity detection of P. nicotianae was successful developed. The optimum amplification of RPA products was observed at 38 ℃ within 30 min without PCR thermal cycler. The primers YPT1F, YPT1R and probe YPT1-LFD-probe for RPA assay were designed based on the YPT1 gene, which showed high specificity to P. nicotianae. The limit of detection of RPA assay was 10 pg·μL-1 genomic DNA, indicating that RPA has an equal sensitivity to that of conventional PCR. The establishment of RPA assay for the detection of P. nicotianae has the characteristics of rapidity, simpleness and practicality, which provides a new method for the rapid detection of P. nicotianae.
Key words:  Phytophthora nicotianae  RPA  specificity  sensitivity

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