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LAMP detection primer composition for phytophthora infestans and LAMP detection kit and LAMP detection method of LAMP detection primer composition

A primer composition and technology of Phytophthora infestans, applied in biochemical equipment and methods, methods based on microorganisms, measurement/inspection of microorganisms, etc., can solve the problems of poor specificity, long cycle and low sensitivity of detection methods, and achieve Strong specificity, high accuracy, and the effect of improving the reaction rate

Active Publication Date: 2015-01-28
瑞测精准医学检测(上海)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Purpose of the invention: In view of the problems in the prior art that the biological detection method of Phytophthora infestans requires a long period, the detection method has poor specificity and low sensitivity, the purpose of the present invention is to provide a LAMP detection primer composition for Phytophthora infestans

Method used

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  • LAMP detection primer composition for phytophthora infestans and LAMP detection kit and LAMP detection method of LAMP detection primer composition
  • LAMP detection primer composition for phytophthora infestans and LAMP detection kit and LAMP detection method of LAMP detection primer composition
  • LAMP detection primer composition for phytophthora infestans and LAMP detection kit and LAMP detection method of LAMP detection primer composition

Examples

Experimental program
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Effect test

Embodiment 1

[0042] A LAMP detection kit for detecting Phytophthora infestans, consisting of 1.6 μM forward inner primer FIP, 1.6 μM reverse inner primer BIP, 0.2 μM forward outer primer F3, 0.2 μM reverse outer primer B3, 0.2 μM Reverse loop primer LB, 1.4mM dNTPs, 20mM Tris-HCl pH 8.8, 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 6mM MgSO 4 , 0.1% Triton X-100, 8U Bst DNA polymerase8 units, 5mM hydroxynaphthol blue, and add ultrapure water to prepare 25uL detection solution. The specific sequences of each primer are as follows:

[0043] FIP: 5′-AATCGCGAAAGCCATGTGAGCCAAACGACCTTTTGTAAGG-3′;

[0044] BIP: 5′-TTGCTTAGAAAATCCGTACGATCGGACAAATGTTTTTTTAGCGGC-3′;

[0045] F3: 5'-TGTGAGTGTCTAACATATTTTACG-3';

[0046] B3: 5'-GTTAGTTAAATAGGAAATCACGC-3';

[0047] LB: 5'-GGCAAGACCATCAAGCTCCAA-3'.

[0048] Wherein, the forward inner primer FIP, the reverse inner primer BIP, the forward outer primer F3, the reverse outer primer B3 and the reverse loop primer LB can directly form a LAMP detection primer compo...

Embodiment 2

[0049] The specificity test of embodiment 2 Phytophthora infestans LAMP reaction

[0050]In order to verify the specificity of the LAMP method, 30 Phytophthora infestans strains, 13 other oomycetes and 19 pathogenic fungi from Jiangsu, Shandong and Fujian provinces were used as test materials. All mold strains can be observed with a blue positive reaction or agarose gel electrophoresis with LAMP ladder-like bands, and the remaining 13 oomycetes and 19 pathogenic fungi have purple negative reactions or no agarose gel electrophoresis. Amplified bands appear. Select different species (P. sojae; Phytophthora parasitica; Phytophthora capsici; Phytophthora strawberry; Phytophthora ramie) and different genera (Pythium ultima; Fusarium equiseti; bacteria; Rhizoctonia solani; Verticillium dahliae) DNA as a template, take 1 μL DNA solution, add 23 μL of the detection solution prepared in Example 1 and 1 μL sterilized deionized water to carry out LAMP reaction, the reaction program is: ...

Embodiment 3

[0051] The sensitivity test of embodiment 3 Phytophthora infestans LAMP reaction

[0052] In order to determine the sensitivity of the LAMP detection method, the extracted DNA of Phytophthora infestans was measured with a spectrophotometer (1 μg / μL), then diluted 10 times with DEPC water, and stored at -70°C as a template. Take 1 μL of the 10-fold diluted DNA dilution solution of each concentration as a template, add 23 μL of the detection solution prepared in Example 1 and 1 μL of sterilized deionized water for LAMP reaction, and the reaction program is: 64°C for 80 min. Get 2 μ L amplified products loading sample, electrophoresis on 2% agarose gel, the result shows that the LAMP method can detect the DNA of Phytophthora infestans that concentration is 1ng; HNB color reaction shows that the sensitivity of LAMP reaction also reaches 1ng ( image 3 with Figure 4 ).

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Abstract

The invention discloses an LAMP detection primer composition for phytophthora infestans and an LAMP detection kit and an LAMP detection method of the LAMP detection primer composition. The LAMP detection primer composition consists of a positive inner primer FIP, a reverse inner primer BIP, a positive outer primer F3, a reverse outer primer B3 and a reverse ring primer LB. The detection method disclosed by the invention is high in accuracy, high in specificity, convenient to operate and high in practicability, realizes constant-temperature multiplication, also provides a new technical platform for detection on the phytophthora infestans and can be used for performing high-sensitivity quick detection on the phytophthora infestans; meanwhile, pathogens can be identified at the initial stage of disease invasion, and the pathogens in field soil can be detected. The LAMP detection primer composition has an important significance for preventing and treating late blight of potatoes and tomatoes, reducing the blind use of pesticide, lowering the production cost and alleviating the environmental pollution of the pesticide.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a LAMP detection primer composition of Phytophthora infestans, a LAMP detection kit and a LAMP detection method thereof. Background technique [0002] Phytophthora infetans is the first species of Phytophthora discovered in the genus Phytophthora, which can cause late blight of potatoes, mainly infecting potatoes, tomatoes and other crops, causing severe yield reduction or even failure. Potato late blight is a devastating oomycete disease that causes potato tuber rot and stem and leaf death. It is a worldwide disease that seriously affects potato yield, quality and industrialization development. In 1847, the large-scale occurrence of this disease caused potato Harvest failures lead to famines. Phytophthora infestans has always been one of the most serious diseases in potato and tomato production. In recent years, potato late blight has been increasing year by year in my ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 戴婷婷郑小波吴小芹
Owner 瑞测精准医学检测(上海)有限公司
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