Anoectochilus formosanus anthracnose LAMP detection primer and visible detection method thereof

A technology for detection primers and detection methods, which is applied in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, etc. Reliable results and highly sensitive results

Inactive Publication Date: 2016-01-06
陈子岩 +3
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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 gold wire for the traditional detection method of A. LAMP detection primers for lotus anthracnose and visual detection method for A. clematis anthracnose with reliable results, easy operation, strong specificity and high sensitivity

Method used

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  • Anoectochilus formosanus anthracnose LAMP detection primer and visible detection method thereof
  • Anoectochilus formosanus anthracnose LAMP detection primer and visible detection method thereof
  • Anoectochilus formosanus anthracnose LAMP detection primer and visible detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Visual detection of LAMP primers for anthracnose of Clematis clematis

[0049] 1. LAMP Visual Detection of Clematis anthracnose

[0050] ①LAMP reaction system: 25μL reaction system containing 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mMKCl, 8mM MgSO 4 , betaine 0.8mol / L, Bst DNA polymerase 8U, dNTPs 1.0mmol / L, F3 and B3 0.2mmol / L, FIP and BIP each 1.6mmol / L, calcein 50μmol / L, manganese chloride 500μmol / L, Tween-200.1%, template DNA50 ~100ng, make up the deficiency with sterile double distilled water; LAMP reaction conditions are incubation at 63-65°C for 45-60min, inactivation at 85°C for 5-10min.

[0051] 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 judged as negative.

[005...

Embodiment 2

[0054] Example 2: Specific amplification of LAMP primers to Clematis anthracnose

[0055] 1. Specific detection of LAMP for anthracnose of Clematis aureus

[0056] ①LAMP reaction system: 25μl reaction system containing 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mMKCl, 8mM MgSO 4 , betaine 0.8mol / L, Bst DNA polymerase 8U, dNTPs 1.0mmol / L, F3 and B3 0.2mmol / L, FIP and BIP each 1.6mmol / L, calcein 50μmol / L, manganese chloride 500μmol / L, Tween-200.1%, template DNA50 ~100ng, make up the deficiency with sterile double distilled water; LAMP reaction conditions are incubation at 63-65°C for 45-60min, inactivation at 85°C for 5-10min.

[0057] 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 judged as negative...

Embodiment 3

[0060] Example 3: Sensitivity detection of LAMP primers to Clematis anthracnose

[0061] 1. Sensitivity detection of LAMP for anthracnose of Clematis aureus

[0062] Using the 10-fold concentration serial dilution method, the extracted DNA of Clematis anthracnose bacteria was diluted to 1000ng, 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 100ag, 10ag, a total of 9 different concentration gradients.

[0063] ①LAMP reaction system: 25μl reaction system containing 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mMKCl, 8mM MgSO 4 , betaine 0.8mol / L, Bst DNA polymerase 8U, dNTPs 1.0mmol / L, F3 and B3 0.2mmol / L, FIP and BIP each 1.6mmol / L, calcein 50μmol / L, manganese chloride 500μmol / L, Tween-200.1%, template DNA50 ~100ng, make up the deficiency with sterile double distilled water; LAMP reaction conditions are incubation at 63-65°C for 45-60min, inactivation at 85°C for 5-10min.

[0064] ②After the LAMP reaction, green fluorescence was observed as a positive color, and orange...

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PUM

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Abstract

The invention discloses an anoectochilus formosanus anthracnose LAMP detection primer and a visible detection method thereof, which are particularly used for the specificity detection of anoectochilus formosanus anthracnose. The anoectochilus formosanus anthracnose LAMP primer is mainly designed, and green fluorescence or a trapezoidal zone having LAMP characteristics can be observed by virtue of the developing reaction after the constant-temperature amplification of LAMP or agarose gel electrophoresis detection. The LAMP primer and the application thereof can be used for rapidly, sensitively and accurately detecting a plant infecting the anoectochilus formosanus anthracnose bacteria in the production practice and can also be used for the early diagnosis of field diseases and monitoring and identification of bacteria, so that a reliable technical and theoretical evidence can be provided for preventing and controlling the anoectochilus formosanus anthracnose.

Description

technical field [0001] The LAMP detection primer for A. clematis anthracnose and its visual detection method of the present invention are specially used for high specificity, sensitivity and visual rapid detection of A. clematis anthracnose, and can be used for early diagnosis of clematis anthracnose and monitoring and identification of pathogens at the same time The invention belongs to the technical field of detection, identification and control of crop diseases. Background technique [0002] Clematis clematis is a perennial rare Chinese herbal medicine, also known as the flower-leaved lip orchid, and some people call it golden silk grass and golden thread to the bone, etc. It has high medicinal value, such as clearing heat and cooling blood, eliminating wind and dampness, Strong heart and diuretic, solid kidney, flat liver and other effects. As a highly respected health-care medicinal material in recent years, merchants have developed many related products, which are wid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/6844C12Q2531/119
Inventor 陈子岩张群林刘小丽李本金
Owner 陈子岩
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