基于URP-PCR多态性片段的苦瓜枯萎病菌特异性检测技术的建立
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摘要
通过URP(Universal Rice Primers)-PCR分析尖孢镰孢菌苦瓜专化型基因组DNA扩增片段多态性,筛选检测尖孢镰孢菌苦瓜专化型的特异性引物,并建立了基于该引物的PCR检测方法。结果表明,特异性引物为FOMM-SPF/FOMMSPR,PCR检测体系为25μL,包括2"Green Taq M aster Mix 12.5μL,10 mmol·L~(-1)的上下游引物各1μL,模板DNA 1μL,灭菌去离子水补足至25μL; PCR程序为95℃预变性3 min,94℃变性15 s,57℃退火30 s,72℃延伸20 s,共30个循环,循环结束后72℃延伸5 min;特异性扩增片段大小294 bp,检测灵敏度为2 ng·μL~(-1)DNA或50个孢子·500 mg~(-1)土壤。该引物及其检测方法对尖孢镰孢菌苦瓜专化型的检测特异性好、灵敏度高,可以从土壤和植物样品中快速准确地检测出苦瓜枯萎病菌,无需病原菌的分离培养和致病性检测,对苦瓜枯萎病的早期诊断和预警及有效防控具有重要的指导意义。
Special primers and detection approach were developed based on the DNA amplification fragment polymorphs by URP( Universal Rice Primers)-PCR in Fusarium oxysporum f. sp. momodicae causing bitter gourd wilt. The application showed that the specific 294 bp amplicon was generated with the specific primer pair FOMM-SPF/FOMM-SPR in 25 μL of detection volume including 12.5 μL of 2 "Green Taq Master Mix,1 μL of 10 μmol·L~(-1) each primer,1μL of template DNA,and sterile ddH_2O to obtain the final volume; Cycling conditions were 3 min initial denaturation at 95℃,30 cycles of 15 s at 94℃,30 s at 57℃,20 s at 72℃ and final elongation 5 min. The specific primer pair for 294 bp product and the detection sensitivity is 2 ng· μL~(-1) for genomic DNA of F. oxysporum f. sp. momodicae and 50 spores·500 mg~(-1) soil for the soil pathogen. The processing methods exhibited the high specificity and sensitivity F. oxysporum f. sp. momodicae that could be detected in the soil and plant samples rapidly and accurately instead of pathogen isolation and pathogenicity test,which would be beneficial in significance for the early diagnosis,early warning,and effective control of the bitter gourd Fusarium wilt disease.
Special primers and detection approach were developed based on the DNA amplification fragment polymorphs by URP( Universal Rice Primers)-PCR in Fusarium oxysporum f. sp. momodicae causing bitter gourd wilt. The application showed that the specific 294 bp amplicon was generated with the specific primer pair FOMM-SPF/FOMM-SPR in 25 μL of detection volume including 12.5 μL of 2 "Green Taq Master Mix,1 μL of 10 μmol·L~(-1) each primer,1μL of template DNA,and sterile ddH_2O to obtain the final volume; Cycling conditions were 3 min initial denaturation at 95℃,30 cycles of 15 s at 94℃,30 s at 57℃,20 s at 72℃ and final elongation 5 min. The specific primer pair for 294 bp product and the detection sensitivity is 2 ng· μL~(-1) for genomic DNA of F. oxysporum f. sp. momodicae and 50 spores·500 mg~(-1) soil for the soil pathogen. The processing methods exhibited the high specificity and sensitivity F. oxysporum f. sp. momodicae that could be detected in the soil and plant samples rapidly and accurately instead of pathogen isolation and pathogenicity test,which would be beneficial in significance for the early diagnosis,early warning,and effective control of the bitter gourd Fusarium wilt disease.
引文
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[16]Lee H H,Kang N,Park I,et al.Characterization of new ly bred cordyceps militaris strains for higher production of cordycepin through HPLC and URP-PCR analysis[J].Journal of Microbiology and Biotechnology,2017,27(7):1223-1232.
[17]SankhLa A K,Ma Lik C P,Parashar M.A review on start codon targeted(SCoT)marker[J].Journal of Plant Science Research,2015,31(2):153-160.
[18]Xiong F,Liu J,Jiang J,et al.Molecular profiling of genetic variability in domesticated groundnut(Arachis hypogaea L.)based on ISJ,URP,and DAM D markers[J].Biochemical Genetics,2013,51:889-900.
[19]Mann M B,Spadari C C,Feltrin T,et al.Genetic variability of bipolaris sorokiniana isolates using URP-PCR[J].Tropical Plant Pathology 2014,39(2):163-171.
[2]Qi P K.A survey on the Fusarium oxysporum f.spp.of Cucurbitaceae(in Chinese)[J].Journal of South China Agricultural University(华南农业大学学报),1995,16(4):110-114.
[3]Zhu T S,Qi P K.Study on the pathogen of bitter gourd wilt in Guangdong(in Chinese)[J].Journal of South China Agricultural University(华南农业大学学报),1998,19(4):14-18.
[4]Zhang S.Studies oil the physiological differentiation and differential expression of FochsV gene from three melons Fusarium wilt(in Chinese)[D].Shenyang:Shenyang Agricultural University(沈阳:沈阳农业大学),2013.
[5]Chen Z D,Yuan G Q,Li Q Q,et al.Identification and genetic diversity of Fusarium oxysporum isolates from bitter gourd(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2014,44(1):36-45.
[6]Wang Q,Lin L.Research progress on Fusarium wilt of cucurbits(in Chinese)[J].China Cucurbits and Vegetables(中国瓜菜),2016,29(3):1-6.
[7]Zhang S M,Zhao X Y,Zhang X C,et al.Molecular detection of Fusarium oxysporum in cucumber,watermelon and melon(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2010,40(6):636-641.
[8]Su J,Gao Z G,Yao Y,et al.Assessment of genetic diversity among Fusarium wilt pathogens of the cucurbits detected by ISSR markers(in Chinese)[J].Northern Horticulture(北方园艺),2014(17):97-101.
[9]Lin Y H,Chen K S,Chang J Y,et al.Development of the molecular methods for rapid detection and differentiation of Fusarium oxysporum and F.oxysporum f.sp.niveum in Taiwan[J].New Biotechnology,2010,27(4):409-418.
[10]Chang P,Lin Y,Chang J,et al.Molecular detection of Fusarium oxysporum f.sp niveum,the causing agent of watermelon Fusarium wilt disease[J].Phytopathology,2009,99(6):S21-S21.
[11]Chen Z D,Huang R K,Li Q Q,et al.Development of pathogenicity and AFLP to characterize Fusarium oxysporum f.sp.momordicae isolates from Bitter gourd in China[J].Journal of Phytopathology,2015,163(3):202-211.
[12]Chen Z D.Studies on the identification,genetic diversity of Fusarium oxysporum f.sp.momodicae and differentially proteomic analysis on the pathogen-host interaction(in Chinese)[D].Guangxi:Guangxi University(广西:广西大学),2014.
[13]Kang H W.Genetic divesity analysis of fungal species by universal rice primer(URP)-PCR[J].Korean Journal of Mycology,2012,40(2):78-85.
[14]Kang H W,Park D S,Park Y J,et al.PCR based detection of Phellinus linteus using specific primers generated from universal rice primer(URP)derived PCRpolymorphic band[J].M ycobiology,2002,30(4):202-207.
[15]Kang H W,Park D S,Go S J,et al.Fingerprinting of diverse genomes using PCR with universal rice primers generated from repetitive sequence of Korean w eedy rice[J].M olecules&Cells,2002,13(2):281-287.
[16]Lee H H,Kang N,Park I,et al.Characterization of new ly bred cordyceps militaris strains for higher production of cordycepin through HPLC and URP-PCR analysis[J].Journal of Microbiology and Biotechnology,2017,27(7):1223-1232.
[17]SankhLa A K,Ma Lik C P,Parashar M.A review on start codon targeted(SCoT)marker[J].Journal of Plant Science Research,2015,31(2):153-160.
[18]Xiong F,Liu J,Jiang J,et al.Molecular profiling of genetic variability in domesticated groundnut(Arachis hypogaea L.)based on ISJ,URP,and DAM D markers[J].Biochemical Genetics,2013,51:889-900.
[19]Mann M B,Spadari C C,Feltrin T,et al.Genetic variability of bipolaris sorokiniana isolates using URP-PCR[J].Tropical Plant Pathology 2014,39(2):163-171.