南京农业大学学报2024，Vol.47Issue(4)：665-673,9.DOI:10.7685/jnau.202305019
尖孢镰刀菌西瓜专化型RPA检测方法的建立与评价
The establishment and evaluation of a specialized RPA detection method for Fusarium oxysporum f.sp.niveum
摘要
Abstract
[Objectives]A recombinase polymerase amplification(RPA)system was constructed for the rapid detection of Fusarium oxysporum f.sp.niveum(FON)in order to provide technical support for the rapid detection of watermelon wilt in the field.[Methods]The genome sequences of 11 pathogenic fungi,including FON,were downloaded from the NCBI website for bioinformatics analysis using SeqHunter 2 software.Four specific gene fragments of FON were obtained by comparison and primers were designed and screened for them.The specificity and sensitivity of the primers were verified using polymerase chain reaction(PCR),and in addition,the specificity and sensitivity of the primers were verified using the established RPA detection system,and finally,the RPA detection evaluation of artificially inoculated watermelon plants was performed.[Results]The RPA detection system for FON was established,and the optimal reaction time(40 min)and optimal reaction temperature(30℃)of the amplification system of DNA constant temperature rapid amplification kit were explored,and the detection limit of the optimal reaction system was 100 pg·μL-1.When using the DNA constant temperature rapid amplification kit(colloidal gold test strip type)for amplification,it only needed to react at 38℃for 12 min,and 50 μL of the amplified product diluted 20 times was detected with the colloidal gold test strip.After 5 min,the results could be judged,and the detection limit was 100 pg·μL-1.[Conclusion]The RPA detection system of FON was successfully constructed.The established RPA detection system could detect FON from the genomic DNA of infected watermelon plant tissues,which could provide technical support for the rapid detection of watermelon Fusarium wilt in the field.关键词
西瓜枯萎病/尖孢镰刀菌西瓜专化型/重组酶聚合酶扩增技术(RPA)/快速检测Key words
watermelon wilt disease/Fusarium oxysporum f.sp.niveum/recombinant enzyme polymerase amplification technology(RPA)/rapid detection分类
农业科技引用本文复制引用
董文盼,杨纪潇,杨项明,吕明慧,朱彦泽,蒋春号..尖孢镰刀菌西瓜专化型RPA检测方法的建立与评价[J].南京农业大学学报,2024,47(4):665-673,9.基金项目
中央高校基本业务费专项资金(KYZZ2022001) （KYZZ2022001）
江苏省重点研发计划项目(BE2021364) （BE2021364）
