Peronophythora litchii LAMP (loop-mediated isothermal amplification) primer and rapid detection method thereof

A technique for detection of Pythophthora litchie and its detection method, which is applied in the field of LAMP primers and rapid detection of Pythora litchie, can solve the problems of poor specificity, long period, and low sensitivity of the detection method, and achieve strong specificity, reliable results, and high sensitivity. high 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

[0005] The purpose of the present invention is to provide a kind of LAMP detection primer of litchi downy mildew and reliable result, easy to solve the problem that the biological detection method of litchi downy mi

Method used

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  • Peronophythora litchii LAMP (loop-mediated isothermal amplification) primer and rapid detection method thereof
  • Peronophythora litchii LAMP (loop-mediated isothermal amplification) primer and rapid detection method thereof
  • Peronophythora litchii LAMP (loop-mediated isothermal amplification) primer and rapid detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: LAMP primer is to the specific amplification of litchi peronosophthora

[0050] 1. Specific detection of LAMP on Lychee peronosophthora

[0051] ①LAMP reaction system: 25.0 μL: including 0.2 μM each of F3 and B3, 1.6 μM each of FIP and BIP, 1× ThermopolBuffer, 1.0 mM dNTPs, 0.8 M betaine, 6.0 mM magnesium sulfate, 0.1% Tween-20,

[0052] 8U Bst The DNA polymerase is 50ng template DNA, 50μM calcein, 500μM manganese chloride, and the insufficient part is made up with sterile double distilled water.

[0053] The LAMP reaction condition is to incubate at 63° C. for 60 minutes, and then incubate at 80° C. for 5 minutes.

[0054] ②After the LAMP reaction is completed, if green fluorescence is observed in the color development results, it is judged as positive, and orange is judged as negative; or take 2 μL of the amplification product and use 2.0% agarose gel electrophoresis to detect it. If the characteristic trapezoidal band of LAMP appears, it is judged...

Embodiment 2

[0057] Embodiment 2: Sensitivity detection of LAMP primers to Lychee peronosophthora

[0058] 1. Sensitivity Detection of LAMP on Lychee Peronosporae

[0059] Using 10-fold concentration serial dilution method, the extracted DNA of Peronospora litchie was diluted to 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, 1fg / μL, 100ag / μL, a total of 8 different concentration gradients.

[0060] ①LAMP reaction system is 25uL: including 0.2μM each of F3 and B3, 1.6μM each of FIP and BIP, 1×ThermopolBuffer, 1.0mMdNTPs, 0.8M betaine, 6.0mM magnesium sulfate, 0.1%Tween-20,

[0061] 8U Bst The DNA polymerase is 1 μL template DNA, 50 μM calcein, 500 μM manganese chloride, and the insufficient part is made up with sterile double distilled water.

[0062] The LAMP reaction condition is to incubate at 63° C. for 60 minutes, and then incubate at 80° C. for 5 minutes.

[0063] ②After the LAMP reaction is completed, if green fluorescence is observed in the color development results, ...

Embodiment 3

[0065] Embodiment 3: detection of litchi downy mildew in pathogenic tissue

[0066] 1. Sample collection: Plant tissue samples were collected from the litchi production base in Fuzhou, Fujian.

[0067] 2. DNA extraction and detection

[0068] The DNA of P. litchi was extracted from the diseased plant tissue by NaOH rapid lysis method.

[0069] Perform LAMP detection as follows:

[0070] ①LAMP reaction system is 25 μL: including 0.2 μM each of F3 and B3, 1.6 μM each of FIP and BIP, 1× ThermopolBuffer, 1.0 mM dNTPs, 0.8 M betaine, 6.0 mM magnesium sulfate, 0.1% Tween-20,

[0071] 8U Bst The DNA polymerase is 1 μL template DNA, 50 μM calcein, 500 μM manganese chloride, and the insufficient part is made up with sterile double distilled water.

[0072] The LAMP reaction condition is to incubate at 63° C. for 60 minutes, and then incubate at 80° C. for 5 minutes.

[0073] ② After the LAMP reaction is completed, if green fluorescence is observed in the color development res...

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Abstract

The invention discloses a peronophythora litchii LAMP (loop-mediated isothermal amplification) primer and a rapid detection method thereof which are specially applied to specific detection of peronophythora litchii. The peronophythora litchii LAMP primer is mainly adopted and designed, and through isothermal amplification visual inspection or agarose gel electrophoresis detection, green fluorescence can be observed or an LAMP characteristic ladder can appear. The LAMP primer and a usage method thereof can be applied to rapid, sensitive and accurate detection of the peronophythora litchii in plants infected with the peronophythora litchii in production practice, can also be applied to early diagnosis of diseases and pests in fields as well as monitoring and identification of germs, and provide reliable technical and theoretical bases for control of diseases and pests caused by the peronophythora litchii.

Description

technical field [0001] The invention relates to a LAMP primer for downy mildew of litchi and a rapid detection method thereof, which is specially used for high-sensitivity rapid molecular detection of downy mildew of litchi and can be used for early diagnosis of downy mildew of litchi in the field and monitoring and identification of pathogens, belonging to crops Disease detection, identification and prevention technology field. Background technique [0002] Litchi downy mildew is a common and serious disease in litchi producing areas in my country, and its pathogen is downy blight of litchi ( peronophythoralitchii ). The disease was first reported in Taiwan, my country in 1978, and it is currently occurring in Guangdong, Guangxi, Fujian, Hainan, Taiwan and other places. and young fruit, causing a large number of flower drop, fruit drop, cracked fruit and rotten fruit, the yield loss is as high as 80%, or even no harvest, which is one of the main reasons for the long-term l...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/645
CPCC12Q1/6895
Inventor 李本金陈庆河刘裴清刘小丽翁启勇
Owner INST OF PLANT PROTECTION FAAS
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