基于环介导等温扩增技术快速检测瓜果腐霉菌
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摘要
[目的]建立一种快速、准确的检测瓜果腐霉菌(Pythium aphanidermatum)的环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,为诊断和防治瓜果腐霉菌引起的植物病害提供技术支撑。[方法]通过比较基因组学方法,在瓜果腐霉菌基因组上筛选有序列特异性的基因作为检测的靶标基因,设计LAMP特异性引物,建立一种基于颜色判定的快速、准确的LAMP检测方法,随后对该检测方法的特异性、灵敏度和发病组织实际检测等进行分析评价。[结果]鉴定到1个编码Trypsin蛋白酶的基因是瓜果腐霉菌特异的基因,以该序列作为靶标建立了LAMP检测方法。在扩增前加入染料羟基萘酚蓝(HNB)作为指示剂,在等温条件(64℃)下进行核酸扩增反应60 min,即可通过肉眼直接观察结果,HNB显示天蓝色即为阳性反应;同时,扩增产物经琼脂糖凝胶电泳验证为梯形条带,也可判定为阳性反应。特异性试验中,建立的LAMP检测方法能将瓜果腐霉菌和其他病原菌区分开。灵敏度试验中,LAMP检测方法的最低检测限为100 pg·μL-1,与Real-time PCR反应灵敏度一样,是普通PCR反应灵敏度的100倍。发病组织实际检测试验中,该LAMP方法能检测出人工接种发病植株中的瓜果腐霉菌。[结论]成功建立了一种适用于基层使用的针对瓜果腐霉菌的快速检测技术,为该菌所致病害的准确诊断奠定了技术基础。
[Objectives]The objective of this study is to establish a rapid and accurate loop-mediated isothermal amplification( LAMP)method for detecting Pythium aphanidermatum,and to provide technical supports for monitoring and prevention of plant diseases caused by P. aphanidermatum. [Methods]A comparative genomics approach was used to identify specific target gene for P. aphanidermatum.The LAMP method was established,and was evaluated for specificity,sensitivity,and detection of inoculated plant tissues. [Results]One gene encoding Trypsin protease was specific for P. aphanidermatum,and was used as the target. The LAMP method efficiently amplified the target gene at 64 ℃ for 60 min,and LAMP products were visualized by adding hydroxynaphthol blue( HNB) before amplification. The amplification products that turned blue were considered to be positive. Meanwhile,the positive DNA products were visualised as a ladder-like banding pattern on 20 g·L-1 gel electrophoresis. The specificity test showed that positive results were only obtained from P. aphanidermatum isolates. The detection limit was 100 pg·μL-1,which was the same with Real-time PCR,and was nearly 100 times higher than that of the conventional PCR. In addition,this LAMP method was successfully applied to detect P. aphanidermatum DNA extracted from inoculated plant tissues. [Conclusions]We successfully established a simple,rapid and accurate LAMP method for detecting P. aphanidermatum in the field.
[Objectives]The objective of this study is to establish a rapid and accurate loop-mediated isothermal amplification( LAMP)method for detecting Pythium aphanidermatum,and to provide technical supports for monitoring and prevention of plant diseases caused by P. aphanidermatum. [Methods]A comparative genomics approach was used to identify specific target gene for P. aphanidermatum.The LAMP method was established,and was evaluated for specificity,sensitivity,and detection of inoculated plant tissues. [Results]One gene encoding Trypsin protease was specific for P. aphanidermatum,and was used as the target. The LAMP method efficiently amplified the target gene at 64 ℃ for 60 min,and LAMP products were visualized by adding hydroxynaphthol blue( HNB) before amplification. The amplification products that turned blue were considered to be positive. Meanwhile,the positive DNA products were visualised as a ladder-like banding pattern on 20 g·L-1 gel electrophoresis. The specificity test showed that positive results were only obtained from P. aphanidermatum isolates. The detection limit was 100 pg·μL-1,which was the same with Real-time PCR,and was nearly 100 times higher than that of the conventional PCR. In addition,this LAMP method was successfully applied to detect P. aphanidermatum DNA extracted from inoculated plant tissues. [Conclusions]We successfully established a simple,rapid and accurate LAMP method for detecting P. aphanidermatum in the field.
引文
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[2]Larkin R P,English J T,Mihail J D.Effects of infection by Pythium spp.on root system morphology of alfalfa seedlings[J].Phytopathology,1995,85(4):430-435.
[3]Hendrix F F,Jr,Campbell W A.Pythiums as plant pathogens[J].Annual Review of Phytopathology,1973,11(1):77-98.
[4]Postma J,Geraats B P J,Pastoor R,et al.Characterization of the microbial community involved in the suppression of Pythium aphanidermatum in cucumber grown on rockwool[J].Phytopathology,2005,95(7):808-818.
[5]Kageyama K,Suzuki M,Priyatmojo A,et al.Characterization and identification of asexual strains of Pythium associated with root rot of rose in Japan[J].Journal of Phytopathology,2003,151(9):485-491.
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[7]郭世保,陈俊华,史洪中.黄瓜苗期主要病害的识别与防治[J].现代农村科技,2015(1):27.Guo S B,Chen J H,Shi H Z.Identification and control of major diseases of cucumber seedling[J].Modern Agricultural Science and Technology,2015(1):27(in Chinese).
[8]潘金菊,于婷,尚玉珂,等.生防菌BMP-11对瓜果腐霉病菌的抑制活性及其鉴定[J].植物保护学报,2008,35(4):311-316.Pan J J,Yu T,Shang Y K,et al.Antifungal activity of antagonistic strain BMP-11 against Pythium aphanidermatum and its identification[J].Acta Phytophylacica Sinica,2008,35(4):311-316(in Chinese with English abstract).
[9]张旭,尚楠,张宝,等.抗瓜果腐霉芽孢杆菌优良菌株的筛选及生物学特性[J].食品科学,2012,33(5):138-143.Zhang X,Shang N,Zhang B,et al.Screening and biological characteristics of Bacillus sp.with high anti-fungal activity against Pythium aphanidermatum[J].Food Science,2012,33(5):138-143(in Chinese with English abstract).
[10]Schroeder K L,Okubara P A,Tambong J T,et al.Identification and quantification of pathogenic Pythium spp.from soils in eastern Washington using real-time polymerase chain reaction[J].Phytopathology,2006,96(6):637-647.
[11]Botton S A,Pereira D I B,Costa M M,et al.Identification of Pythium insidiosum by nested PCR in cutaneous lesions of Brazilian horses and rabbits[J].Current Microbiology,2011,62(4):1225-1229.
[12]Cullen D W,Toth I K,Boonham N,et al.Development and validation of conventional and quantitative polymerase chain reaction assays for the detection of storage rot potato pathogens,Phytophthora erythroseptica,Pythium ultimum and Phoma foveata[J].Journal of Phytopathology,2007,155(5):309-315.
[13]Morisset D,Stebih D,Cankar K,et al.Alternative DNA amplification methods to PCR and their application in GMO detection:a review[J].European Food Research and Technology,2008,227(5):1287-1297.
[14]Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12):e63.
[15]Goto M,Honda E,Ogura A,et al.Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue[J].Biotechniques,2009,46(3):167-172.
[16]Fukuta S,Kato S,Yoshida K,et al.Detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction[J].Journal of Virological Methods,2003,112:35-40.
[17]Dai T T,Lu C C,Lu J,et al.Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae[J].FEMS Microbiology Letters,2012,334(1):27-34.
[18]Rigano L A,Marano M R,Castagnaro A P,et al.Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods[J].BMC Microbiology,2010,10(1):176-184.
[19]Fukuta S,Takahashi R,Kuroyanagi S,et al.Development of loop-mediated isothermal amplification assay for the detection of Pythium myriotylum[J].Letters in Applied Microbiology,2014,59(1):49-57.
[20]Takahashi R,Fukuta S,Kuroyanagi S,et al.Development and application of a loop-mediated isothermal amplification assay for rapid detection of Pythium helicoides[J].FEMS Microbiology Letters,2014,355(1):28-35.
[21]郑小波.疫霉菌及其研究技术[M].北京:中国农业出版社,1997:81-82.Zheng X B.Phytophthora and Its Research Technology[M].Beijing:China Agriculture Press,1997:81-82(in Chinese).
[22]Fukuta S,Takahashi R,Kuroyanagi S,et al.Detection of Pythium aphanidermatum in tomato using loop-mediated isothermal amplification(LAMP)with species-specific primers[J].European Journal of Plant Pathology,2013,136(4):689-701.
[23]戴婷婷,陆辰晨,沈浩,等.基于环介导等温扩增技术检测橡树疫霉菌[J].南京农业大学学报,2013,36(3):23-28.DOI:10.7685/j.issn.1000-2030.2013.03.004.Dai T T,Lu C C,Shen H,et al.Rapid diagnostic methods for Phytophthora ramorum using LAMP[J].Journal of Nanjing Agricultural University,2013,36(3):23-28(in Chinese with English abstract).
[24]沈浩,戴婷婷,吴翠萍,等.基于环介导等温扩增技术检测大豆北方茎溃疡病菌[J].南京农业大学学报,2015,38(2):255-260.DOI:10.7685/j.issn.1000-2030.2015.02.012.Shen H,Dai T T,Wu C P,et al.The tef1α-LAMP method for rapid detection of Diaporthe phaseolorum var.caulivora[J].Journal of Nanjing Agricultural University,2015,38(2):255-260(in Chinese with English abstract).
[25]戴婷婷,陆辰晨,郑小波.环介导等温扩增技术在病原物检测上的应用研究进展[J].南京农业大学学报,2015,38(5):695-703.DOI:10.7685/j.issn.1000-2030.2015.05.001.Dai T T,Lu C C,Zheng X B.Application research progress of Loop-mediated isothermal amplification in the pathogenic microorganism[J].Journal of Nanjing Agricultural University,2015,38(5):695-703(in Chinese with English abstract).
[26]Belkhiri A,Buchko J,Klassen G R.The 5S ribosomal RNA gene in Pythium species:two different genomic locations[J].Molecular Biology and Evolution,1992,9(6):1089-1102.
[27]Buchko J,Klassen G R.Detection of length heterogeneity in the ribosomal DNA of Pythium ultimum by PCR amplification of the intergenic region[J].Current Genetics,1990,18(3):203-205.
[28]Wang P H,Wang Y T,White J G.Species-specific PCR primers for Pythium developed from ribosomal ITS1 region[J].Letters in Applied Microbiology,2003,37(2):127-132.
[2]Larkin R P,English J T,Mihail J D.Effects of infection by Pythium spp.on root system morphology of alfalfa seedlings[J].Phytopathology,1995,85(4):430-435.
[3]Hendrix F F,Jr,Campbell W A.Pythiums as plant pathogens[J].Annual Review of Phytopathology,1973,11(1):77-98.
[4]Postma J,Geraats B P J,Pastoor R,et al.Characterization of the microbial community involved in the suppression of Pythium aphanidermatum in cucumber grown on rockwool[J].Phytopathology,2005,95(7):808-818.
[5]Kageyama K,Suzuki M,Priyatmojo A,et al.Characterization and identification of asexual strains of Pythium associated with root rot of rose in Japan[J].Journal of Phytopathology,2003,151(9):485-491.
[6]王晓鸣,吴全安,刘晓娟,等.寄生玉米的6种腐霉及其致病性研究[J].植物病理学报,1994(4):343-346.Wang X M,Wu Q A,Liu X J,et al.Identification and pathogenicity of Pythium spp.isolated from maize[J].Acta Phytopathologica Sinica,1994(4):343-346(in Chinese with English abstract).
[7]郭世保,陈俊华,史洪中.黄瓜苗期主要病害的识别与防治[J].现代农村科技,2015(1):27.Guo S B,Chen J H,Shi H Z.Identification and control of major diseases of cucumber seedling[J].Modern Agricultural Science and Technology,2015(1):27(in Chinese).
[8]潘金菊,于婷,尚玉珂,等.生防菌BMP-11对瓜果腐霉病菌的抑制活性及其鉴定[J].植物保护学报,2008,35(4):311-316.Pan J J,Yu T,Shang Y K,et al.Antifungal activity of antagonistic strain BMP-11 against Pythium aphanidermatum and its identification[J].Acta Phytophylacica Sinica,2008,35(4):311-316(in Chinese with English abstract).
[9]张旭,尚楠,张宝,等.抗瓜果腐霉芽孢杆菌优良菌株的筛选及生物学特性[J].食品科学,2012,33(5):138-143.Zhang X,Shang N,Zhang B,et al.Screening and biological characteristics of Bacillus sp.with high anti-fungal activity against Pythium aphanidermatum[J].Food Science,2012,33(5):138-143(in Chinese with English abstract).
[10]Schroeder K L,Okubara P A,Tambong J T,et al.Identification and quantification of pathogenic Pythium spp.from soils in eastern Washington using real-time polymerase chain reaction[J].Phytopathology,2006,96(6):637-647.
[11]Botton S A,Pereira D I B,Costa M M,et al.Identification of Pythium insidiosum by nested PCR in cutaneous lesions of Brazilian horses and rabbits[J].Current Microbiology,2011,62(4):1225-1229.
[12]Cullen D W,Toth I K,Boonham N,et al.Development and validation of conventional and quantitative polymerase chain reaction assays for the detection of storage rot potato pathogens,Phytophthora erythroseptica,Pythium ultimum and Phoma foveata[J].Journal of Phytopathology,2007,155(5):309-315.
[13]Morisset D,Stebih D,Cankar K,et al.Alternative DNA amplification methods to PCR and their application in GMO detection:a review[J].European Food Research and Technology,2008,227(5):1287-1297.
[14]Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12):e63.
[15]Goto M,Honda E,Ogura A,et al.Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue[J].Biotechniques,2009,46(3):167-172.
[16]Fukuta S,Kato S,Yoshida K,et al.Detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction[J].Journal of Virological Methods,2003,112:35-40.
[17]Dai T T,Lu C C,Lu J,et al.Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae[J].FEMS Microbiology Letters,2012,334(1):27-34.
[18]Rigano L A,Marano M R,Castagnaro A P,et al.Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods[J].BMC Microbiology,2010,10(1):176-184.
[19]Fukuta S,Takahashi R,Kuroyanagi S,et al.Development of loop-mediated isothermal amplification assay for the detection of Pythium myriotylum[J].Letters in Applied Microbiology,2014,59(1):49-57.
[20]Takahashi R,Fukuta S,Kuroyanagi S,et al.Development and application of a loop-mediated isothermal amplification assay for rapid detection of Pythium helicoides[J].FEMS Microbiology Letters,2014,355(1):28-35.
[21]郑小波.疫霉菌及其研究技术[M].北京:中国农业出版社,1997:81-82.Zheng X B.Phytophthora and Its Research Technology[M].Beijing:China Agriculture Press,1997:81-82(in Chinese).
[22]Fukuta S,Takahashi R,Kuroyanagi S,et al.Detection of Pythium aphanidermatum in tomato using loop-mediated isothermal amplification(LAMP)with species-specific primers[J].European Journal of Plant Pathology,2013,136(4):689-701.
[23]戴婷婷,陆辰晨,沈浩,等.基于环介导等温扩增技术检测橡树疫霉菌[J].南京农业大学学报,2013,36(3):23-28.DOI:10.7685/j.issn.1000-2030.2013.03.004.Dai T T,Lu C C,Shen H,et al.Rapid diagnostic methods for Phytophthora ramorum using LAMP[J].Journal of Nanjing Agricultural University,2013,36(3):23-28(in Chinese with English abstract).
[24]沈浩,戴婷婷,吴翠萍,等.基于环介导等温扩增技术检测大豆北方茎溃疡病菌[J].南京农业大学学报,2015,38(2):255-260.DOI:10.7685/j.issn.1000-2030.2015.02.012.Shen H,Dai T T,Wu C P,et al.The tef1α-LAMP method for rapid detection of Diaporthe phaseolorum var.caulivora[J].Journal of Nanjing Agricultural University,2015,38(2):255-260(in Chinese with English abstract).
[25]戴婷婷,陆辰晨,郑小波.环介导等温扩增技术在病原物检测上的应用研究进展[J].南京农业大学学报,2015,38(5):695-703.DOI:10.7685/j.issn.1000-2030.2015.05.001.Dai T T,Lu C C,Zheng X B.Application research progress of Loop-mediated isothermal amplification in the pathogenic microorganism[J].Journal of Nanjing Agricultural University,2015,38(5):695-703(in Chinese with English abstract).
[26]Belkhiri A,Buchko J,Klassen G R.The 5S ribosomal RNA gene in Pythium species:two different genomic locations[J].Molecular Biology and Evolution,1992,9(6):1089-1102.
[27]Buchko J,Klassen G R.Detection of length heterogeneity in the ribosomal DNA of Pythium ultimum by PCR amplification of the intergenic region[J].Current Genetics,1990,18(3):203-205.
[28]Wang P H,Wang Y T,White J G.Species-specific PCR primers for Pythium developed from ribosomal ITS1 region[J].Letters in Applied Microbiology,2003,37(2):127-132.