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A kind of molecular detection primer and detection method of downy mildew of litchi

A technique for molecular detection of downy mildew of litchi, applied in the field of molecular detection primers and detection of downy mildew of litchi, can solve the problems of time-consuming and inability to achieve rapid detection, and achieve simple and fast operation, good practicability, and strong practicability Effect

Active Publication Date: 2018-10-30
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 the problem that the traditional detection method of downy mildew of litchi has high requirements on the experimental skills and practical experience of the operator in the prior art, and is very time-consuming, and cannot achieve rapid inspection. Specific molecular detection primers and a rapid molecular detection system and procedure for Lychee peronospora with reliable results, easy operation, strong specificity and high sensitivity

Method used

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  • A kind of molecular detection primer and detection method of downy mildew of litchi
  • A kind of molecular detection primer and detection method of downy mildew of litchi
  • A kind of molecular detection primer and detection method of downy mildew of litchi

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Primer is to the specific amplification of litchi peronosophthora

[0036] 1. Specific Detection of Peronophytophthora in Litchi

[0037] PCR reaction system 25.0μL, including 2× Taq PCR Master Mix 12.5 μL, 10 μmol / L primers (PvYF1 / PvYR1) 0.5 μL each, DNA template 25ng, the insufficient part was prepared by dd H 2 O make up. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 1 min, annealing at 59°C for 30 sec, extension at 72°C for 1 min, a total of 28 cycles; extension at 72°C for 10 min.

[0038] 2. Test results

[0039] Specificity of detection: In addition to the 249bp product that can be amplified specifically from the DNA of P. litchi peronospora from Fujian, Guangdong, Guangxi, Taiwan and other provinces in my country, the DNA of 13 different oomycetes and 8 kinds of fungi failed to amplify. Increase any product, with strong specificity.

Embodiment 2

[0040] Embodiment 2: Sensitivity detection of primers to downy mildew of litchi

[0041] 1. Dilution of DNA concentration: the extracted Genomic DNA of Pythophthora litchie was measured by a spectrophotometer and then diluted in series.

[0042] 2. Sensitivity detection of downy mildew on litchi

[0043] The extracted P. litchi DNA was sequentially diluted to 10 ng / µL by 10-fold concentration serial dilution method, 1

[0044] ng / µL, 100 pg / µL, 10 pg / µL, 1 pg / µL, 100 fg / µL, 10 fg / µL, 1 fg / µL in 8 different concentration gradients, and the sensitivity of the primers was determined by nested PCR . Take 1 μL of Genomic DNA of Phytophthora litchie at different concentrations as a template, and use Phytophthora universal primers (Yph1F / Yph2R) for the first round of PCR amplification. The reaction system is 25.0 μL, including 2× Taq PCR Master Mix 12.5 μL, 10 μmol / L primers (Yph1F / Yph2R) 1.0 μL each, DNA template 1 μL, insufficient part was prepared by dd H 2 O make up. The...

Embodiment 3

[0046] Example 3: Detection of Peronosophthora lychee in diseased plant samples.

[0047] 1. Sample collection: Plant tissue samples were collected from the litchi production base in Fujian Province.

[0048] 2. DNA extraction and detection

[0049] The diseased plant tissue was extracted by NaOH rapid lysis method, and PCR amplification was performed according to the following method:

[0050] PCR reaction system 25.0μL, including 2× Taq PCR Master Mix 12.5 μL, 10 μmol / L primers (PvYF1 / PvYR1) 0.5 μL each, DNA template 1 μL, the insufficient part was prepared by dd H 2 O make up. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 1 min, annealing at 59°C for 30 sec, extension at 72°C for 1 min, a total of 28 cycles; extension at 72°C for 10 min.

[0051] Amplified products were detected by agarose gel electrophoresis.

[0052] 3. Test results

[0053] see results image 3 , a clear specific band with a molecula...

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Abstract

The invention discloses peronophythora litchii molecular detection primers and a detection method thereof, and is dedicated to specific molecular detection of peronophythora litchi. A pair of peronophythora litchii specific primers PvYF1:5'-GTCCGAGTTTCTAGCAGATTG-3' and PvYR1:5'-ACGATAATACCGTGAGCGCC-3' are mainly designed; through PCR amplification and agarose gel electrophoresis, a specific amplification product with the fragment length of 249 bp is specifically amplified in peronophythora litchii pure DNA and peronophythora litchii-carried plants. The specific molecular detection primers and a usage method thereof can be used for rapid, sensitive and specific detection of peronophythora in the peronophythora litchii infected plants in production practice, at the same time, can be used for early diagnosis of field diseases and monitoring and identification of pathogenic bacteria, and provides a reliable technology and theoretical basis for diseases caused by peronophythora litchii.

Description

technical field [0001] The invention discloses a primer for molecular detection of downy mildew of litchi and a 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 and monitoring and identification of pathogens in the field, and belongs to the detection of crop diseases , identification and prevention technology. Background technique [0002] Litchi is delicious, beautiful in color and rich in nutrition. It is a fruit in tropical and subtropical regions. It is distributed throughout Hainan, Fujian, Guangxi, Guangdong, Taiwan and other provinces, and has important economic value. Phytophthora lychee ( Peronophythora litchii ) is one of the most important diseases of litchi, which seriously affects the yield and quality of litchi, as well as the storage, transportation and export of fresh fruit. The disease was first discovered in Taiwan Provin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12R1/645
CPCC12Q1/6895
Inventor 李本金陈庆河刘裴清刘小丽黄宏臻翁启勇
Owner INST OF PLANT PROTECTION FAAS
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