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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  • Claims
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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 det

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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Example Embodiment

[0041] Example 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, 8 units of 8U Bst DNA polymerase, 5mM hydroxynaphthol blue, add ultrapure water to prepare 25uL detection solution. The specific sequence of each primer is 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] Among them, 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 comp...

Example Embodiment

[0049] Example 2 Specificity test of LAMP reaction of Phytophthora infestans

[0050] In order to verify the specificity of the LAMP method, 30 strains of Phytophthora infestans, 13 other oomycetes and 19 pathogenic fungi from Jiangsu, Shandong and Fujian provinces in my country were used as test materials. The LAMP test results showed 30 pathogenic diseases. The mildew strains can be observed in the sky blue positive reaction or the agarose gel electrophoresis shows the LAMP ladder-like band, the remaining 13 kinds of oomycetes and 19 kinds of pathogenic fungi show a purple negative reaction or the agarose gel electrophoresis does not Amplified bands appear. Select different species from Phytophthora infestans (Phytophthora sojae; Phytophthora parasitica; Phytophthora capsici; Phytophthora strawberry; Phytophthora ramie) and bacteria of different genera (Pythium ultimum; Fusarium equisetum; Colletotrichum grisea; Blast Rhizoctonia solani; Verticillium dahliae) DNA as a template, ...

Example Embodiment

[0051] Example 3 Sensitivity test of LAMP reaction of Phytophthora infestans

[0052] In order to determine the sensitivity of the LAMP detection method, the extracted DNA of Phytophthora infestans was measured with a spectrophotometer (1μg / μL), then diluted with DEPC water 10 times, and stored at -70℃ as a template. Take 1 μL of the DNA diluent of each concentration diluted by 10 times as a template, and add 23 μL of the detection solution prepared in Example 1 and 1 μL of sterilized deionized water for LAMP reaction. The reaction procedure is: 64°C for 80 minutes. Take 2μL of the amplified product and load it on a 2% agarose gel. The results show that the LAMP method can detect the DNA of Phytophthora infestans at a concentration of 1ng; the HNB color reaction shows that the sensitivity of the LAMP reaction is also up to 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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