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LAMP detection primer composition for phytophthotacactorum, as well as LAMP detection kit and LAMP detection method of LAMP detection primer composition

A primer composition and Phytophthora victoria technology, applied in the direction of biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of long period, low sensitivity, and poor specificity of detection methods, and achieve accurate High performance, improved reaction rate, and convenient operation

Active Publication Date: 2015-02-25
瑞测精准医学检测(上海)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 in the prior art, the purpose of the present invention is to provide a LAMP detection primer composition for Phytophthora

Method used

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  • LAMP detection primer composition for phytophthotacactorum, as well as LAMP detection kit and LAMP detection method of LAMP detection primer composition
  • LAMP detection primer composition for phytophthotacactorum, as well as LAMP detection kit and LAMP detection method of LAMP detection primer composition
  • LAMP detection primer composition for phytophthotacactorum, as well as LAMP detection kit and LAMP detection method of LAMP detection primer composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] A LAMP detection kit for detecting Phytophthora victoria, 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, 1.25mM dNTPs, 20mM Tris-HCl pH 8.8, 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 6mM MgSO 4 , 0.1% Triton X-100, Bst DNA polymerase 16 units, 5mM hydroxynaphthol blue, add ultrapure water to prepare 25uL detection solution. The specific sequences of each primer are as follows:

[0040] FIP: 5'-TCTGGGCACAACCGCAAAAA-TTTGCGAGCTCCAGATTTCC-3';

[0041] BIP: 5′-AATCCGTACGATCGAGCTGGAC-ACACGCCACGTCTGCT-3′;

[0042] F3: 5'-TTCTGCGCTAGGCGACC-3';

[0043] B3: 5'-CACACAAGTGGACCGTTAG-3';

[0044] LB: 5'-GGAAAGACCATCAAGCTCCAGAT-3'.

[0045] 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 for detectin...

Embodiment 2

[0046] The specificity test of embodiment 2 phytophthora LAMP reaction

[0047] In order to verify the specificity of the LAMP method, 15 Phytophthora strains, 13 other oomycetes and 19 pathogenic fungi were used as test materials (Table 1). positive reaction or agarose gel electrophoresis with LAMP ladder-like bands, and the remaining 13 oomycetes and 19 pathogenic fungi showed purple negative reactions or no amplified bands in agarose gel electrophoresis. Select different species of Phytophthora ramie; Phytophthora camphor; Phytophthora capsici; Plague bacterium; Rhizoctonia solani; Verticillium dahliae; Pythium ultima) DNA as a template, get 1 μL DNA solution, add 23 μL of the detection solution prepared in Example 1 and 1 μL sterilized deionized water to carry out the LAMP reaction, the reaction The program is: 64°C 60min. Based on the agarose gel electrophoresis and color reaction of the reaction system as the result judgment standard, when the DNA template of P.tentacu...

Embodiment 3

[0048] Example 3 Sensitivity Test of Phytophthora victoria LAMP Reaction

[0049] In order to determine the sensitivity of the LAMP detection method, the DNA concentration of the extracted Phytophthora viridans was measured with a spectrophotometer (1 μg / μL), and then diluted 10 times with DEPC water, and stored at -70°C as a template. Take 1 μL of 10-fold diluted DNA dilutions 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 60 min. Get 2 μ L amplified products loading sample, the result shows that agarose gel electrophoresis and HNB chromogenic reaction show that the sensitivity of LAMP reaction reaches the DNA of 100pg Phytophthora viridans ( figure 2 ).

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Abstract

The invention discloses an LAMP detection primer composition for phytophthotacactorum, as well as 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 loop primer LB; detailed sequences of all the primers are as follows: FIP: 5'-TCTGGGCACAACCGCAAAAA-TTTGCGAGCTCCAGATTTCC-3'; BIP: 5'-AATCCGTACGATCGAGCTGGAC-ACACGCCACGTCTGCT-3'; F3: 5'-TTCTGCGCTAGGCGACC-3'; B3: 5'-CACACAAGTGGACCGTTAG3'; LB: 5'-GGAAAGACCATCAAGCTCCAGAT-3'. The detection method disclosed by the invention is high in accuracy, high in specificity, convenient to operate and good in practicability; constant-temperature amplification is realized; meanwhile, a novel technology platform is provided for detection of the phytophthotacactorum; the detection method can be applied to high-sensitivity rapid detection of the phytophthotacactorum; simultaneously, pathogens can be identified in initial stage of invasion of diseases, and the phytophthotacactorum soil of a field can be detected. The LAMP detection primer composition for the phytophthotacactorum, as well as the LAMP detection kit and the LAMP detection method of the LAMP detection primer composition, disclosed by the invention, play an important role in epidemic treatment caused by the phytophthotacactorum, reduction of sightless usage of pesticides, reduction of production cost and reduction of environmental pollution caused by the pesticides as well.

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 viridans, a LAMP detection kit and a LAMP detection method thereof. Background technique [0002] The plant disease caused by Phytophthora cactorum infection is a worldwide distribution of soil-borne and devastating diseases, which harms more than 200 kinds of plants and often affects the growth of various economic crops (such as strawberries, apples, pears, ginseng, etc.). Production suffered serious losses. Phytophthora belongs to the phylum Oomycota, class Oomycetes, order Peronosporales, family Pythiaceae, and genus Phytophthora. The plant diseases caused by Phytophthora are widely distributed, so the establishment of rapid molecular detection technology of Phytophthora is of great significance for the early control of the diseases caused by Phytophthora. At present, there is no quick and effective control ...

Claims

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Application Information

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