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A detection target pcin100006 of Phytophthora camphora and its special detection primer and rapid detection method

A Phytophthora and target technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of long cycle, low sensitivity, and poor specificity of detection methods, and achieve high accuracy, Improved reaction rate, high specificity and high sensitivity

Active Publication Date: 2019-08-20
NANJING FORESTRY UNIV
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  • Description
  • Claims
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Problems solved by technology

[0006] Purpose of the invention: Aiming at the problems of long period, poor specificity and low sensitivity of the biological detection method of Phytophthora camphora in the prior art, the purpose of the present invention is to provide a new detection target Pcinn100006 of Phytophthora camphora and its LAMP Detection primer composition

Method used

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  • A detection target pcin100006 of Phytophthora camphora and its special detection primer and rapid detection method
  • A detection target pcin100006 of Phytophthora camphora and its special detection primer and rapid detection method
  • A detection target pcin100006 of Phytophthora camphora and its special detection primer and rapid detection method

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Experimental program
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Effect test

Embodiment 1

[0035] More than 1,000 Phytophthora camphora-specific genes were obtained through whole-genome sequence alignment, and a high-reliability specific molecular detection target Pcinn100006 was excavated from it. Its DNA sequence is shown in SEQ ID NO.1, and its protein sequence is shown in SEQ ID NO. .2 shown. Based on this new target, a sensitive and accurate LAMP detection technology system was established.

[0036] The LAMP detection primer composition used in the LAMP detection technology system: consists of forward inner primer FIP, reverse inner primer BIP, forward outer primer F3, reverse outer primer B3, forward loop primer LF and reverse loop primer LB Composition; each primer sequence is as follows:

[0037] FIP: 5'-CGAAGGACGAGGTGAAGGTGGACGCCCATACATCACATACG-3';

[0038] BIP: 5′-AAGGCCGGCTACATGTACTCGTCTCGGGCAAGATGACTTC-3′;

[0039] F3: 5'-GTTCTGCGCGCGATTTGGTTAG-3';

[0040] B3: 5'-GCGGATCTTCCGATCTGGTA-3';

[0041] LF: 5'-AACGTGACGCCGGCAACAA-3';

[0042] LB: 5'-TGTT...

Embodiment 2

[0048] In order to verify the specific primer sequence of Phytophthora camphora, the present embodiment uses 10 Phytophthora camphora bacterial strains and 14 kinds of other oomycetes and 17 kinds of pathogenic fungi as test materials (Table 1), and adopts the CTAB method to extract Phytophthora camphora in pathogenic tissues. The DNA of the mold. The specific method is as follows: Take a small amount of mycelium powder, add 900 μL 2% CTAB extract and 90 μL 10% SDS, vortex and mix well, put in a water bath at 55°C for 1 hour, and invert several times every 10 minutes. Centrifuge at 12000rpm for 10min, take the supernatant and add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), mix by inverting, centrifuge at 12000rpm for 10min; transfer the supernatant to a new tube, add an equal volume of chloroform, and mix gently by inverting Evenly, centrifuge at 12000rpm for 5min. Transfer the supernatant to a new tube, add 2 times the volume of absolute ethanol and 1 / 10 ...

Embodiment 3

[0054] The sensitivity test of embodiment 3 Phytophthora camphora LAMP reaction and PCR reaction

[0055] In order to determine the sensitivity of the LAMP detection method, the extracted DNA of Phytophthora camphora 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, the result shows that agarose gel electrophoresis and HNB color reaction show that the sensitivity of LAMP reaction reaches the DNA of Phytophthora camphora of 100pg ( figure 2 a). The genomic DNA of the standard strain of Phytophthora camphora with the same concentration was used as the amplification template to carry out PCR amplification reactio...

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Abstract

The invention discloses a phytophthora cinnamomic detection target Pcin100006 and a special detection primer and a fast detection method thereof. The DNA sequence of the detection target Pcin100006 isshown as SEQ ID NO.1. The special detection primer consists of a positive inner primer FIP, a reverse inner primer BIP, a positive outer primer F3, a revere outer primer B3, a positive ring primer LFand a reverse ring primer LB. The detection method provided by the invention has the advantages that the accuracy is high; the specificity is high; the operation is convenient; the practicability ishigh; the constant temperature amplification is realized; meanwhile, a novel technical platform is provided for the phytophthora cinnamomi detection; the detection target can be used for the high-sensitivity fast detection of the phytophthora cinnamomi; meanwhile, the pathogen is identified in the disease infection primary stage; the phytophthora cinnamomi in the field soil can be detected. The diseases caused by phytophthora cinnamomi can be prevented and treated; the blind use of pesticides can be reduced; the production cost is reduced; important significance is also realized in reduction of the environment pollution due to pesticides.

Description

technical field [0001] The invention belongs to the technical field of gene detection of Phytophthora camphora, and specifically relates to a new detection target Pcin100006 of Phytophthora camphora, a rapid LAMP detection primer composition of Phytophthora camphora, a LAMP detection kit and a LAMP detection method thereof. Background technique [0002] Phytophthora cinnamomi belongs to Oomycota, Oomycetes, Peronosporales, Pythiaceae, and Phytophthora. Phytophthora camphora can infect Cinnamomum genus plants and cause diseases such as root rot and branch canker, which will cause a devastating blow to Cinnamomum camphora plants. Phytophthora camphora has a wide range of hosts and can infect thousands of plants. According to reports, Phytophthora camphora has caused changes in forest structure and plant populations in Southwest Australia, and has seriously threatened the local natural ecosystem and biodiversity. At present, there are no effective control measures for the dis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11
Inventor 戴婷婷焦彬彬胡涛徐月
Owner NANJING FORESTRY UNIV
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