Primers for detecting phytophthora cinnamomi and nested PCR detection method

A technology of Phytophthora camphora and Phytophthora, which is applied in the field of crop disease detection, identification and prevention, can solve the problems of low accuracy, cumbersome procedures, and long time consumption, and achieve the effects of high accuracy, good repeatability, and easy operation

Active Publication Date: 2017-06-20
INST OF PLANT PROTECTION FAAS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a kind of primer and nested PCR detection method for detecting Phytophthora camphora, mainly based on morphological characteristics for the detection and identification of Phytophthora camphora in the prior art, the method takes a long time, the program is loaded down with trivial details, experience It is difficult to monitor the occurrence of diseases in time and control the spread and epidemic of pathogenic bacteria, and the difference between rDNA-ITS sequences in existing molecular detection is very small, and it is difficult for some primers designed with ITS as the target The technical deficiency of distinguishing pathogenic bacteria of closely related species provides Phytophthora camphora specific PCR detection primer pair and its detection method, and utilizes the PCR detection primer pair and detection method described in the present invention to detect Phytophthora camphora with high accuracy and specificity Strong, sensitive, easy to operate, short detection time and reliable results

Method used

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  • Primers for detecting phytophthora cinnamomi and nested PCR detection method
  • Primers for detecting phytophthora cinnamomi and nested PCR detection method
  • Primers for detecting phytophthora cinnamomi and nested PCR detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Design of primer pairs for PCR detection of Phytophthora camphora and verification of specificity of primer pairs

[0030] 1. Extraction of Genomic DNA from Phytophthora camphora

[0031] The CTAB method was used to extract the genomic DNA of Phytophthora camphora from different regions. The specific method was as follows: Take a small amount of mycelium powder in a 1.5 mL centrifuge tube (it is better that the mycelium powder just covers the bottom of the semicircle), add 900 µL of 2% CTAB ( cetyltrimethylammonium bromide) extract (2% CTAB; 100 mmol / L Tris-HCl, pH 8.0; 20 mmol / L EDTA, pH 8.0; 1.4mol / L NaCl) and 90 µL SDS ( Sodium dodecylbenzenesulfonate) [Note: CTAB and SDS need to be preheated at 60°C], use a shaker to shake and mix, 60°C water bath for 1h (DNA is released into the buffer), 12000 r min -1 Centrifuge for 15 min; take 700 µL of the supernatant, add an equal volume of phenol, chloroform, isoamyl alcohol mixture (volume ratio 25:24:1), shake g...

Embodiment 2

[0040] Embodiment 2: Sensitivity determination of Phytophthora camphora nested PCR detection method

[0041] 1. Conventional PCR amplification

[0042] Genomic DNA of Phytophthora camphora was diluted with sterile ultrapure water, and prepared into series concentrations of 10-fold order of magnitude for future use. Use primer PCINF / PCINR described in the present invention to carry out PCR amplification to the genomic DNA of different serial concentrations, evaluate the sensitivity of this primer to Phytophthora camphora genomic DNA detection, amplification reaction system and reaction procedure are as follows: PCR reaction system 25 μ L, Includes 2 x Taq PCR Master Mix (Beijing Tiangen Biochemical Technology Co., Ltd.) 12.5 µL, 10 µmol / L PCINF / PCINR primers 1.0 µL each, DNA template 1.0 µL, make up to 25 µL with sterile ultrapure water. The amplification reaction program was: pre-denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 1 min, annealing at 59°C...

Embodiment 3

[0048] Example 3: Detection of Phytophthora camphora in the root tissue of blueberry root rot

[0049] Extraction of genomic DNA from diseased root tissue: the diseased root tissue of blueberry root rot caused by Phytophthora camphora infection has been identified by conventional morphology and molecular biology as the sample to be tested, and the DNA is extracted by NaOH rapid lysis method. The specific process As follows: Add 10 µL of 0.5 mol / L NaOH to each mg of plant tissue, thoroughly grind the tissue into a paste in a mortar, transfer it to a 1.5 mL centrifuge tube, centrifuge at 12,000 rpm for 6 min, take 5 µL of the supernatant and add it to 495 µL 0.1 mol / L Tris-HCl (pH=8.0) was mixed evenly, and 1.0 µL was used as a PCR template for amplification.

[0050] Nested PCR amplification detection: using the mentioned DNA as a template, using Ypt1 Gene universal primers ph1F / ph2R (ph1F: 5'-CGACCATTGGCGTGGACTTT-3', Yph2R: 5'-ACGTTC- TCGCAGGCGTATCT-3') for the first round o...

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Abstract

The invention provides primers for detecting phytophthora cinnamomi and a nested PCR detection method. The primer group comprises two pairs of primers, wherein the first pair of primers is universal primers ph1F / Yph2R for the phytophthora Ypt1 gene, and the second pair of primers is specific primers PCINF / PCINR for identifying phytophthora cinnamomi. If the concentration reaches 10fg / [mu] L pathogenic bacteria trace DNA, phytophthora cinnamomi can be accurately detected, the nested PCR detection method for detecting phytophthora cinnamomi has the advantages that the accuracy is high, the specificity is strong, the sensitivity is high, and the detection process is simple, convenient and rapid to operate, the nested PCR detection method can be used for the early diagnosis of diseases caused by phytophthora cinnamomi and the monitoring and identifying for pathogenic bacteria, and the problem that the traditional detecting and identifying methods have complicated steps and long periods is solved.

Description

technical field [0001] The invention provides a primer for detecting Phytophthora camphora, and also provides a nested PCR amplification detection method using the primer set, belonging to the technical field of crop disease detection, identification and prevention. Background technique [0002] Phytophthora camphora ( Phytophthora cinnamomi ) belonging to the Oomycota ( Oomycota ), Oomycetes ( Oomycetes ), Peronomyces ( Peronosporals ), Pythium ( Pythiaceae ), Phytophthora ( Phytophthora ), is an important plant pathogen, widely distributed in Europe, America, Southeast Asia, Oceania and other regions. Phytophthora camphora mainly infects the roots of plants, blocking the transportation of water and nutrients, thus causing the host plant to become diseased. The common symptoms of plant diseases caused by it include withered and yellow leaves that are easy to fall off, trunk ulcers accompanied by exudation of jelly, Stem ulcer (can cause sudden plant death), fruit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12Q1/04
CPCC12Q1/686C12Q1/6895C12Q2549/119C12Q2565/125
Inventor 兰成忠姚锦爱阮宏椿吴玮
Owner INST OF PLANT PROTECTION FAAS
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