尖孢镰刀菌西瓜专化型RPA检测方法的建立与评价OA北大核心CSTPCD

[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.