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Alternaria solani loop-mediated amplification detection primer and detection method thereof

A technology for detection of early blight bacteria and primers, applied in the field of crop disease detection, identification and prevention, can solve the problems of long cycle, poor specificity and low sensitivity of detection methods, and achieve reliable results, strong specificity and high sensitivity

Active Publication Date: 2018-11-13
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The purpose of the present invention is to provide a potato early blight for the traditional detection method of potato early blight in the prior art, which requires a long period, poor specificity of the detection method, PCR detection needs to rely on amplification equipment and other equipment, and low sensitivity. Loop-mediated amplification detection primers for bacteria and a visual detection method for potato early blight bacteria with reliable results, easy operation, strong specificity and high sensitivity

Method used

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  • Alternaria solani loop-mediated amplification detection primer and detection method thereof
  • Alternaria solani loop-mediated amplification detection primer and detection method thereof
  • Alternaria solani loop-mediated amplification detection primer and detection method thereof

Examples

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Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Rapid Detection of Potato Early Blight with Loop-Mediated Amplification Primers

[0048] 1. Loop-Mediated Amplification Rapid Detection of Potato Early Blight

[0049] ①Ring-mediated amplification reaction system: 25μl reaction system containing 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mM KCl, 8mM MgSO 4 , betaine 0.8 mol / L, Bst DNA polymerase 8 U, dNTPs 1.0 mmol / L, F3 and B3 0.2 mmol / L, FIP and BIP each 1.6 mmol / L, calcein 50 μmol / L, manganese chloride 500 μmol / L, TWeen-20 0.1 %, 50ng of template DNA, and make up the deficiency with sterile double-distilled water; the conditions of the loop-mediated amplification reaction were incubation at 64°C for 50 minutes and inactivation at 85°C for 10 minutes.

[0050] ② After the loop-mediated amplification reaction, green fluorescence was observed as a positive result of the color development, and orange was judged as a negative. Alternatively, take 2 μl of the amplification product and test it with 2% agaros...

Embodiment 2

[0053] Example 2: Specific Amplification of Loop-mediated Amplification Primers to Potato Early Blight

[0054] 1. Loop-Mediated Amplification-Specific Detection of Potato Early Blight

[0055] ①Ring-mediated amplification reaction system: 25μl reaction system containing 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mM KCl, 8mM MgSO 4 , betaine 0.8 mol / L, Bst DNA polymerase 8 U, dNTPs 1.0 mmol / L, F3 and B3 0.2 mmol / L, FIP and BIP each 1.6 mmol / L, calcein 50 μmol / L, manganese chloride 500 μmol / L, TWeen-20 0.1 %, 50ng of template DNA, and make up the deficiency with sterile double-distilled water; the conditions of the loop-mediated amplification reaction were incubation at 64°C for 50 minutes and inactivation at 85°C for 10 minutes.

[0056] ② After the loop-mediated amplification reaction, green fluorescence was observed as a positive result of the color development, and orange was judged as a negative. Alternatively, take 2 μl of the amplification product and test it with 2...

Embodiment 3

[0059] Example 3: Sensitivity detection of loop-mediated amplification primers to Potato blight blight

[0060] 1. Sensitive Detection of Loop-Mediated Amplification of Potato Early Blight

[0061] Using the 10-fold concentration serial dilution method, the extracted DNA of Potato blight bacteria was diluted to a concentration of 1: 1.36×10 2 ng / μL; 2: 1.36×10 1 ng / μL; 3: 1.36 ng / μL; 4: 1.36×10 -1 ng / μL; 5: 1.36×10 -2 ng / μL; 6: 1.36×10 -3 ng / μL; 7: 1.36×10 -4 ng / μL;, a total of 7 different concentration gradients.

[0062] ① Loop-mediated amplification reaction system: 25μl reaction system containing 20mM Tris-HCl, 10mM (NH 4 )2 SO 4 , 10mM KCl, 8mM MgSO 4 , betaine 0.8 mol / L, Bst 8 U of DNA polymerase, 1.0 mmol / L of dNTPs, 0.2 mmol / L of F3 and B3, 1.6 mmol / L of FIP and BIP, 50 μmol / L of calcein, 500 μmol / L of manganese chloride, TWeen-200.1% , 50 ng of template DNA, and make up the deficiency with sterile double-distilled water; the conditions of the loop-me...

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Abstract

The invention discloses an alternaria solani loop-mediated amplification detection primer and a detection method thereof. The primer and the detection method are special for the specific detection ofthe alternaria solani. Alternaria solani loop-mediated amplification primers F3, B3, FIP and BIP are mainly used; after the color reaction or agarose gel electrophoresis detection is conducted after loop-mediated isothermal amplification, green fluorescence or a loop-mediated amplification characteristic trapezoidal band can be observed. The loop-mediated amplification primers and the usage can beused for rapid sensitive accurate detection of plants infected with the alternaria solani in production practices, and can also be used for early diagnosis of field diseases and monitoring and identification of pathogens to provide reliable technical and theoretical basis for prevention and control of the alternaria solani.

Description

technical field [0001] The present invention is a ring-mediated amplification detection primer for potato early blight and its detection method, which is specially used for rapid detection of potato early blight with high specificity and sensitivity, and can be used for early diagnosis of potato early blight and monitoring and identification of the pathogen at the same time The invention belongs to the technical field of detection, identification and control of crop diseases. Background technique [0002] Alternaria solani ( Alternaria solani ) caused by early blight is an important disease in potato production. It occurs and prevails in all major potato producing areas in the world, and it seriously threatens potato production. It causes great harm and is difficult to control. Early blight is widespread and serious in our country, and has become an obstacle to limit the potato production and industrialization in our country. Due to the lack of effective early diagnosis te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 陈庆河迈赫兰王荣波李本金刘裴清翁启勇
Owner INST OF PLANT PROTECTION FAAS
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