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Phytophthora vignae LAMP (loop-mediated isothermal amplification) detection primers and phytophthora vignae LAMP detection method

A technology for detection primers and detection methods, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of long cycle, poor specificity and low sensitivity of detection methods, and achieve reliable results, strong specificity, Sensitive effect

Active Publication Date: 2016-02-17
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a kind of Phytophthora cowpea in the prior art for the traditional detection method of Phytophthora cowpea required period is long, detection method specificity is poor, PCR detection needs to rely on equipment such as amplification instrument, and sensitivity is low, provides a kind of Phytophthora cowpea LAMP detection primers and a detection method with reliable results, easy operation, strong specificity and high sensitivity

Method used

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  • Phytophthora vignae LAMP (loop-mediated isothermal amplification) detection primers and phytophthora vignae LAMP detection method
  • Phytophthora vignae LAMP (loop-mediated isothermal amplification) detection primers and phytophthora vignae LAMP detection method

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Effect test

Embodiment 1

[0042] Example 1: Specific amplification of LAMP primers to Phytophthora cowpea

[0043] 1. LAMP Specific Detection of Phytophthora vignacea

[0044] ①LAMP reaction system: In the 25μl reaction system, the concentration of F3 and B3 primers is 0.2mmol / L, the concentration of FIP and BIP primers is 1.6mmol / L, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mMKCl, 8mM MgSO 4 , betaine 0.8mol / L, Bst Polymerase 8U, dNTPs 1.0mmol / L, calcein 50μmol / L, manganese chloride 500μmol / L, Tween-200.1%, template DNA 50ng, make up the deficiency with sterile double distilled water; Incubate for 50 minutes, inactivate at 85°C for 10 minutes.

[0045] 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 2

[0048] Embodiment 2: Sensitivity detection of LAMP primers to Phytophthora cowpea

[0049] 1. LAMP Sensitive Detection of Phytophthora vignacea

[0050] The extracted Phytophthora cowpea DNA was diluted to 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg / μL by 10-fold concentration serial dilution method, a total of 9 different concentration gradients.

[0051] ①LAMP reaction system: In the 25μl reaction system, the concentration of F3 and B3 primers is 0.2mmol / L, the concentration of FIP and BIP primers is 1.6mmol / L, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mMKCl, 8mM MgSO 4 , betaine 0.8mol / L, Bst Polymerase 8U, dNTPs 1.0mmol / L, calcein 50μmol / L, manganese chloride 500μmol / L, Tween-200.1%, template DNA 100ng, make up the deficiency with sterile double distilled water; Incubate for 60 minutes, inactivate at 85°C for 5 minutes.

[0052] ②After the LAMP reaction, green fluorescence was observed as a positive color, and orange was negative. Or take 2 μl of the ampli...

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Abstract

The invention discloses phytophthora vignae LAMP (loop-mediated isothermal amplification) detection primers and a phytophthora vignae LAMP detection method which are specially applied to specific detection of phytophthora vignae. The phytophthora vignae LAMP primers are mainly adopted and designed, and green fluorescence can be observed or an LAMP characteristic ladder can appear through the chromogenic reaction or agarose gel electrophoresis detection after LAMP. The LAMP primers and a usage method thereof can be applied to rapid, sensitive and accurate detection of the cowpea diseases in production practice, can also be applied to early diagnosis of diseases in fields as well as monitoring and identification of germs, and provide reliable technical and theoretical bases for control of the cowpea diseases.

Description

technical field [0001] The primers for detection of Phytophthora cowpea LAMP and the detection method thereof of the present invention are specially used for high-sensitivity and fast LAMP detection of Phytophthora cowpea, and can be used for early diagnosis of cowpea blight in the field and monitoring and identification of pathogens, and belong to the detection, identification and control technology of crop diseases. field. Background technique [0002] by Phytophthora vigna ( Phytophthoravignae ) infection of cowpea blight is the main disease of cowpea adult plants, the suitable temperature for onset is 25-28°C, and with the increase of rainwater and humidity, the disease will become more serious, so the period of continuous cloudy rain and plum rain in spring is the high incidence of disease Expect. Secondly, if the ventilation is not good, the drainage is poor, the application of immature basal fertilizer or continuous cropping, the disease will be more serious. The...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 陈庆河翁启勇李本金刘裴清刘小丽
Owner INST OF PLANT PROTECTION FAAS
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