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A kind of lamp detection primer composition of Phytophthora tunnerii and its lamp detection kit and lamp detection method

A technology of Phytophthora kuhnii and primer composition, which is applied in biochemical equipment and methods, microbe determination/testing, DNA/RNA fragments, etc., can solve the problems of poor specificity, low sensitivity, and long cycle of detection methods, and achieve Easy operation, improved reaction rate and high accuracy

Active Publication Date: 2017-01-18
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: Aiming at the problems of long period, poor specificity and low sensitivity of the biological detection method of Phytophthora kuveii in the prior art, the purpose of the present invention is to provide a LAMP detection primer composition for Phytophthora kuveii

Method used

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  • A kind of lamp detection primer composition of Phytophthora tunnerii and its lamp detection kit and lamp detection method
  • A kind of lamp detection primer composition of Phytophthora tunnerii and its lamp detection kit and lamp detection method
  • A kind of lamp detection primer composition of Phytophthora tunnerii and its lamp detection kit and lamp detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] A LAMP detection kit for the detection of Phytophthora kuhnii, 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, 1.8mM dNTPs, 20mM Tris-HCl pH 8.8, 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 6mM MgSO 4 , 0.1% Triton X-100, BstDNA polymerase 16 units, 5mM hydroxynaphthol blue, add ultrapure water to prepare 25uL detection solution. The specific sequences of each primer are as follows:

[0041] FIP: 5'-CGGATTTTCTAGAACGTGGTACCAA-AATGAAGAGTCGACTCTAGCA-3';

[0042] BIP: 5′-ACTATTGAGCTGGACGGCAA-TCGATAGCAGCCCAAGAG-3′;

[0043] F3: 5'-GTGATCCTTTCACCCTGG-3';

[0044] B3: 5'-TTACAAATGTCAGCTGGATG-3';

[0045] LB: 5'-TGTACGTCTACAGAGGATTTGGAT-3'.

[0046] 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 composition ...

Embodiment 2

[0047] The specificity test of embodiment 2 Phytophthora kucheni LAMP reaction

[0048] In order to verify the specificity of the LAMP method, 8 strains of Phytophthora kundii and 14 kinds of other oomycetes and 17 kinds of pathogenic fungi were used as test materials (Table 1). The LAMP detection results showed that all 8 strains of Phytophthora Positive reaction to sky blue or agarose gel electrophoresis with LAMP ladder-like bands, and purple negative reaction for the remaining 14 oomycetes and 17 pathogenic fungi or no amplified bands in agarose gel electrophoresis . Select different species (Phytophthora sojae; Phytophthora parasitic; Phytophthora infestans; Phytophthora xylica; Phytophthora strawberry; Phytophthora ramie, etc.) Magnaporthe oryzae; Rhizoctonia solani; Verticillium dahliae; Pythium ultima) DNA as a template, get 1 μL of DNA solution, add 23 μL of the detection solution prepared in Example 1 and 1 μL of sterilized deionized water to carry out the LAMP reac...

Embodiment 3

[0049] Example 3 Sensitivity Test of Phytophthora konkeri LAMP Reaction

[0050]In order to determine the sensitivity of the LAMP detection method, the DNA concentration (1 μg / μL) of the extracted Phytophthora kunkleri was measured with a spectrophotometer, and 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, the result shows agarose gel electrophoresis and HNB chromogenic reaction show that the sensitivity of LAMP reaction reaches the DNA of Phytophthora kunklerii of 1ng ( image 3 ).

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Abstract

The invention discloses an LAMP detection primer composition for detecting phytophthora drechsler tucker, an LAMP detection kit and an LAMP detection method. The LAMP detection primer composition comprises a forward inner primer FIP, a backward inner primer BIP, a forward outer primer F3, a backward outer primer B3 and a backward loop primer. The detection method disclosed by the invention is high in accuracy, high in specificity, convenient to operate and good in practicality, achieves isothermal amplification, provides a new technical platform for detecting phytophthora drechsler tucker, is used for high-sensitivity fast detection of phytophthora drechsler tucker and the identification of pathogens at the initial stage of disease infection, and can be used to detect phytophthora drechsler tucker in field soil. The LAMP detection primer composition for detecting phytophthora drechsler tucker, the LAMP detection kit and the LAMP detection method disclosed by the invention have significance of disease control caused by phytophthora drechsler tucker and reducing of blind use of pesticides, can reduce production cost, and has the great importance in reducing environmental pollution caused by the pesticides.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a LAMP detection primer composition of Phytophthora kuveii, a LAMP detection kit and a LAMP detection method thereof. Background technique [0002] Phytophthora drechsleri was first isolated from potato tubers by Drechsler in 1931. Phytophthora subternii was reported in my country in 1982, a new species that can infect cucumbers. At present, the plant blight caused by Phytophthora kuerii infection is a worldwide distribution of soil-borne and destructive diseases, which harms a wide range of hosts and often brings serious serious problems to the production of various economic crops (such as cucumber, sugar beet, potato, cedar, etc.). loss. Phytophthora spp. belongs to the phylum Oomycota, the class Oomycetes, the order Peronosporales, the family Pythiaceae, and the genus Phytophthora. The plant diseases caused by Phytophthora kuerii are widely distributed, so t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 戴婷婷叶建仁吴小芹陈宏健
Owner 瑞测精准医学检测(上海)有限公司
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