LAMP (Loop-Mediated Isothermal Amplification) primer of phytophthora nicotianae and fast detection method thereof

A technology for Phytophthora nicotiana and its detection method, which is applied in the field of Phytophthora tobacco LAMP primers and its rapid detection, can solve the problems of poor specificity, long cycle, and low sensitivity of the detection method, and achieve strong specificity, reliable results, and high sensitivity Effect

Active Publication Date: 2014-05-07
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to provide a kind of LAMP detection primer of Phytophthora tabacum and reliable result, easy operation, Rapid detection method of Phytophthora tobacco with strong specificity and high sensitivity

Method used

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  • LAMP (Loop-Mediated Isothermal Amplification) primer of phytophthora nicotianae and fast detection method thereof
  • LAMP (Loop-Mediated Isothermal Amplification) primer of phytophthora nicotianae and fast detection method thereof
  • LAMP (Loop-Mediated Isothermal Amplification) primer of phytophthora nicotianae and fast detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Specific amplification of Phytophthora nicotiana with LAMP primers

[0043] 1. LAMP Specific Detection of Phytophthora Tobacco

[0044] ①LAMP reaction system 25 μl: containing 0.2 μM each of F3 and B3, 1.6 μM each of FIP and BIP, 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10mM KCl, 8mM MgSO 4 , 0.1% Tween-20, 0.8M Betaine, 1.4 mM dNTPs, 8U Bst Large fragment of DNA polymerase, 25ng of DNA template, make up the deficiency with sterile double distilled water; the LAMP reaction conditions are incubation at 65°C for 60 minutes, and incubation at 82°C for 10 minutes.

[0045] ② Add 1 μl of chromogen to the final amplification product of the LAMP reaction. The chromogen is SYBR green I. If the color development results are observed, the green fluorescence is judged as positive, and the orange is judged as 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 appea...

Embodiment 2

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

[0049] 1. LAMP Sensitivity Detection of Phytophthora Tobacco

[0050] The extracted DNA of Phytophthora nicotiana was diluted into nine different concentration gradients of 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, 100 ag, 10ag and 1ag by 10-fold concentration serial dilution method.

[0051] ①LAMP reaction system 25μl: containing 0.2μM each of F3 and B3, 1.6μM each of FIP and BIP, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mM KCl, 8mM MgSO 4 , 0.1% Tween-20, 0.8M Betaine, 1.4 mM dNTPs, 8U Bst Large fragment of DNA polymerase, 25ng of DNA template, make up the deficiency with sterile double distilled water; the LAMP reaction conditions are incubation at 65°C for 60 minutes, and incubation at 82°C for 10 minutes.

[0052] ② Add 1 μl of chromogen to the final amplification product of the LAMP reaction. The chromogen is SYBR green I. If the color development results are observed, the gree...

Embodiment 3

[0054] Example 3: Detection of Phytophthora nicotiana in diseased tissue or soil.

[0055] 1. Sample collection: Plant tissue and soil samples were collected from the tobacco production base in Nanping, Fujian.

[0056] 2. DNA extraction and detection

[0057] Phytophthora nicotiana DNA was extracted from diseased plant tissue by NaOH rapid cracking method, and Phytophthora nicotiana DNA was extracted by soil DNA extraction method from diseased soil.

[0058] Perform LAMP detection as follows:

[0059]①LAMP reaction system 25 μl: containing 0.2 μM each of F3 and B3, 1.6 μM each of FIP and BIP, 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10mM KCl, 8mM MgSO 4 , 0.1% Tween-20, 0.8M Betaine, 1.4 mM dNTPs, 8U Bst Large fragment of DNA polymerase, 25ng of DNA template, make up the deficiency with sterile double distilled water; the LAMP reaction conditions are incubation at 65°C for 60 minutes, and incubation at 82°C for 10 minutes.

[0060] ② Add 1 μl of chromogen to the fin...

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Abstract

The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) primer of phytophthora nicotianae and a fast detection method thereof, which are especially used for specific detection of the phytophthora nicotianae. The LAMP primer adopted and designed is F3, B3, FIP and BIP, and the sequence of the LAMP primer is shown in SEQIDNO.1-4. Green fluorescence can be observed or the ladder pattern of LAMP characteristic occurs by isothermal amplification and addition of SYBRgreenI color-developing agent for color development or sepharose for gel-electrophoresis detection. The LAMP primer and the use method can be used for fast, sensitive and accurate detection of the phytophthora nicotianae in plants and soil infected by the phytophthora nicotianae in production practice, simultaneously can be used for early diagnosis of diseases in the field and monitoring and identification of germs, and provide reliable technical and theoretical basis for controlling the diseases caused by the phytophthora nicotianae.

Description

technical field [0001] The invention relates to a Phytophthora nicotiana LAMP primer and a rapid detection method thereof, which is specially used for high-sensitivity and rapid molecular detection of Phytophthora tobacco, and can be used for the early diagnosis of tobacco blight in the field and the monitoring and identification of pathogens, belonging to the detection, identification and identification of crop diseases. Prevention technology field. Background technique [0002] by Phytophthora tabacum ( Phytophthora nicotianae Tobacco black shank caused by Breda de Haan is a devastating worldwide disease. In 1896, van Breda de Haan was first discovered in Java, Indonesia. Since then, the disease has spread rapidly. In 1922, it became a serious disease in Florida and Kansas in the United States. After 1924, the disease has spread all over the tobacco fields in temperate, subtropical and tropical regions of the world. It was first reported in China in 1950. At that time, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/04C12Q1/6844C12Q2531/119
Inventor 陈庆河李本金刘裴清谢世勇翁启勇
Owner INST OF PLANT PROTECTION FAAS
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