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A kind of litchi peronosophthora lamp primer and its rapid detection method

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

Active Publication Date: 2018-11-09
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 mildew needs long period, detection method specificity is poor, and sensitivity is low in the prior art. A rapid detection method for Lychee downy mildew with strong operation, strong specificity and high sensitivity

Method used

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  • A kind of litchi peronosophthora lamp primer and its rapid detection method
  • A kind of litchi peronosophthora lamp primer and its rapid detection method
  • A kind of litchi peronosophthora lamp primer and its rapid detection method

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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 is 25.0µL: including 0.2µM each of F3 and B3, 1.6µM each of FIP and BIP, 1×Thermopol Buffer, 1.0 mM dNTPs, 0.8 M betaine, 6.0 mM magnesium sulfate, 0.1% Tween-20,

[0052] 8U Bst DNA polymerase is 50 ng template DNA, 50 μM calcein, 500 μM 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 as...

Embodiment 2

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

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

[0059] The extracted P. litchi DNA was diluted to 1 ng / µL, 100 pg / µL, 10 pg / µL, 1 pg / µL, 100 fg / µL, 10 fg / µL, 1 fg / µL by 10-fold concentration serial dilution method. There are 8 different concentration gradients of μL and 100ag / μL.

[0060] ①LAMP reaction system is 25 uL: 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,

[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 res...

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 resul...

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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 ( peronophythora litchii ). 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12N15/11C12R1/645
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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