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A kind of detection primer of potato infestans lamp and its visual detection method

A potato late blight and detection primer technology, which is applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., can solve the problems of poor specificity, low sensitivity and long period of detection methods, and achieves strong specificity and high sensitivity. , reliable results

Active Publication Date: 2021-04-09
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The purpose of the present invention is to provide a kind of potato infestans in the prior art for the traditional detection method of potato infestans infestation that the required period is long, the specificity of the detection method is poor, PCR detection needs to rely on equipment such as amplification instrument, and the sensitivity is low, provide a kind of LAMP detection primers for P. infestans and a visual detection method for P. infestans with reliable results, easy operation, strong specificity and high sensitivity

Method used

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  • A kind of detection primer of potato infestans lamp and its visual detection method
  • A kind of detection primer of potato infestans lamp and its visual detection method
  • A kind of detection primer of potato infestans lamp and its visual detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Visual detection of LAMP primers for Pseudomonas infestans

[0048] 1. LAMP Visual Detection of Potato Phytophthora infestans

[0049] ①LAMP 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 %, 50-100 ng of template DNA, and make up the deficiency with sterile double-distilled water; the LAMP reaction conditions are incubation at 63-65°C for 45-60min, and inactivation at 85°C for 5-10min.

[0050] ②After the LAMP reaction, green fluorescence was observed as a positive color, and orange was negative. Alternatively, take 2 μl of the amplification product and use 2% agarose gel electrophoresis to detect it. If the characteristic trapezoidal band of LAMP appears, it is judged as positive, and if no amplification band appears...

Embodiment 2

[0053] Example 2: Specific amplification of LAMP primers to P. infestans

[0054] 1. LAMP Specific Detection of Potato Phytophthora infestans

[0055] ①LAMP 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 %, 50-100 ng of template DNA, and make up the deficiency with sterile double-distilled water; the LAMP reaction conditions are incubation at 63-65°C for 45-60min, and inactivation at 85°C for 5-10min.

[0056] ②After the LAMP reaction, green fluorescence was observed as a positive color, and orange was negative. Alternatively, take 2 μl of the amplification product and use 2% agarose gel electrophoresis to detect it. If the characteristic trapezoidal band of LAMP appears, it is judged as positive, and if no amplification band appears, ...

Embodiment 3

[0059] Example 3: Sensitivity detection of LAMP primers to P. infestans

[0060] 1. LAMP Sensitive Detection of Potato Phytophthora infestans

[0061] Using the 10-fold concentration serial dilution method, the extracted DNA of P. infestans was diluted to a concentration of 1:1.28×10 2 ng / μL; 2: 1.28×10 1 ng / μL; 3: 1.28 ng / μL; 4: 1.28×10 -1 ng / μL; 5: 1.28×10 -2 ng / μL; 6: 1.28×10 -3 ng / μL; 7: 1.28×10 -4 ng / μL;, a total of 7 different concentration gradients.

[0062] ①LAMP 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 %, 50-100 ng of template DNA, and make up the deficiency with sterile double-distilled water; the LAMP reaction conditions are incubation at 63-65°C for 45-60min, and inactivation at 85°C for 5-10min.

[0063] ②...

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Abstract

The invention discloses a LAMP detection primer for potato infestans and a visual detection method thereof, which are specially used for specific detection of potato infestans. The LAMP primers F3, B3, FIP, and BIP of a potato infestans were mainly designed. After the chromogenic reaction or agarose gel electrophoresis detection after the constant temperature amplification of LAMP, green fluorescence or the characteristic ladder-shaped band of LAMP can be observed. The invented LAMP primers and their usage can be used for rapid, sensitive and accurate detection of plants infected by P. Provide reliable technical and theoretical basis for the prevention and treatment of the disease.

Description

technical field [0001] The invention discloses a LAMP detection primer for potato infestans and its visual detection method, which is specially used for high-specificity, sensitive and visual rapid detection of potato infestans, and can be used for early diagnosis of potato infestans in the field and monitoring and identification of pathogens, belonging to Crop disease detection, identification and control technology field. Background technique [0002] by Phytophthora infestans ( Phytophthora infestans Late blight caused by (Mont) de Bary) is a devastating disease in potato production. It occurs and is prevalent in all major potato producing areas in the world, and seriously threatens potato production. The damage it causes, the difficulty of control and the The social impact has surpassed rice blast and wheat stripe rust, and is regarded as the largest crop disease in the world. Late blight is common and serious in our country, causing an economic loss of about 8 billion...

Claims

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Application Information

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