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2015•Hebei Nongye Daxue xuebaoRequires access

Development of a loop-mediated isothermal amplification assay for detection of Clavibactermichiganensissubsp.michiganensis

Zhao Sa

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Abstract

A visual loop-mediated isothermal amplification(LAMP)detection of bacterial canker and wilt of tomato was developed with SYBR Green I as a fluorescence indicator in this study.Based on the special internal transcribed spacer(ITS)sequence of Cmm,a set of four LAMP primers was designed.The final reaction system is:template DNA 200 ng,the optimum concentration of MgSO4 was 3.0mmol/L,Bst DNA polymerase 2U,optimal concentration of dNTPs for 0.3mmol/L,and the ratio of outer primers and internal primers was 1∶3for optimum amplification in the total 25μL reaction system.The results showed that the constant temperature of 65 ℃for 40 min could acquire good amplification results.The detection sensitivity of LAMP was 50CFU/mL by naked eyes detection after SYBR Green I dying,theprimers were high specific for Cmm detecting.The LAMP assay developed in this study was simple,fast,sensitive and specific,with good practicability.

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What this paper is about

A visual loop-mediated isothermal amplification(LAMP)detection of bacterial canker and wilt of tomato was developed with SYBR Green I as a fluorescence indicator in this study.Based on the special internal transcribed spacer(ITS)sequence of Cmm,a set of four LAMP primers was designed.The final reaction system is:template DNA 200 ng,the optimum concentration of MgSO4 was 3.0mmol/L,Bst DNA polymerase 2U,optimal concentration of dNTPs for 0.3mmol/L,and the ratio of outer primers and internal primers was 1∶3for optimum amplification in the total 25μL reaction system.The results showed that the constant temperature of 65 ℃for 40 min could acquire good amplification results.The detection sensitivity of LAMP was 50CFU/mL by naked eyes detection after SYBR Green I dying,theprimers were high specific for Cmm detecting.The LAMP assay developed in this study was simple,fast,sensitive and specific,with good practicability.

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Available abstract

A visual loop-mediated isothermal amplification(LAMP)detection of bacterial canker and wilt of tomato was developed with SYBR Green I as a fluorescence indicator in this study.Based on the special internal transcribed spacer(ITS)sequence of Cmm,a set of four LAMP primers was designed.The final reaction system is:template DNA 200 ng,the optimum concentration of MgSO4 was 3.0mmol/L,Bst DNA polymerase 2U,optimal concentration of dNTPs for 0.3mmol/L,and the ratio of outer primers and internal primers was 1∶3for optimum amplification in the total 25μL reaction system.The results showed that the constant temperature of 65 ℃for 40 min could acquire good amplification results.The detection sensitivity of LAMP was 50CFU/mL by naked eyes detection after SYBR Green I dying,theprimers were high specific for Cmm detecting.The LAMP assay developed in this study was simple,fast,sensitive and specific,with good practicability.

Key concepts: Loop-mediated isothermal amplification, SYBR Green I, Molecular biology, Polymerase chain reaction, DNA, Recombinase Polymerase Amplification, Chemistry, Chromatography

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Development of a loop-mediated isothermal amplification assay for detection of Clavibactermichiganensissubsp.michiganensis — Research Paper | ScholarLens