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Phyrophthora infestans LAMP detection primer and visual detection method thereof

A technology for potato late blight and detection primers, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of long cycle, low sensitivity, and poor specificity of detection methods, and achieve reliable results. The effect of high sensitivity and strong specificity

Active Publication Date: 2017-07-28
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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  • Phyrophthora infestans LAMP detection primer and visual detection method thereof
  • Phyrophthora infestans LAMP detection primer and visual detection method thereof
  • Phyrophthora infestans LAMP detection primer and visual detection method thereof

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 45-60 min, and inactivation at 85°C for 5-10 min.

[0050] ②After the LAMP reaction, green fluorescence was observed as a positive color, and orange was negative. Or 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, it is ju...

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 45-60 min, and inactivation at 85°C for 5-10 min.

[0056] ②After the LAMP reaction, green fluorescence was observed as a positive color, and orange was negative. Or 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, it is judg...

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 P. infestans DNA 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] ②Af...

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Abstract

The invention discloses a phyrophthora infestans LAMP detection primer and a visual detection method thereof, and the phyrophthora infestans LAMP detection primer is specially used for specific detection of phyrophthora infestans. The phyrophthora infestans LAMP primer F3, B3, FIP and BIP is mainly designed. Green fluorescence or a ladder pattern with the LAMP characteristic feature can be observed through LAMP chromogenic reaction after isothermal amplification or agarose gel electrophoresis detection. The LAMP primer and the using method thereof can be used for fast, sensitively and accurately detecting plants infected by phyrophthora infestans, can be used for monitoring and authenticating of infestans and early-stage diagnosis of field disease damage and provides reliable technical and theoretical basis for prevention and control of potato late blight.

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 billi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12Q1/04
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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