LAMP detection primer combination of eugenopsis and LAMP detecting kit and method thereof

A technology of Phytophthora syringae and composition, which is applied in the field of LAMP detection primer composition of Phytophthora syringae, can solve the problems of long period, low sensitivity, and poor specificity of detection methods, and achieve high accuracy, convenient operation, specificity and The effect of high sensitivity

Inactive Publication Date: 2018-09-28
NANJING FORESTRY UNIV
View PDF5 Cites 2 Cited by
  • Summary
  • 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 syringae in the prior art, the purpose of the present invention is to provide a ring-mediated isothermal amplification detection primer for Phytophthora syringae combination

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • LAMP detection primer combination of eugenopsis and LAMP detecting kit and method thereof
  • LAMP detection primer combination of eugenopsis and LAMP detecting kit and method thereof
  • LAMP detection primer combination of eugenopsis and LAMP detecting kit and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] A loop-mediated isothermal amplification detection kit for detecting Phytophthora syringae, consisting of 1.6 μM forward inner primer FIP, 1.6 μM reverse inner primer BIP, 0.2 μM forward outer primer F3, and 0.2 μM reverse outer primer B3, 1.8mM dNTPs, 20mM Tris-HCl pH8.8, 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 6mM MgSO 4 , 0.1% Triton X-100, BstDNApolymerase 16 units, 5mM hydroxynaphthol blue, and add ultrapure water to prepare 25uL detection solution. The specific sequences of each primer are as follows:

[0035] FIP: 5'-GCCGCGGTAGTAGCTGCTAGT-TTGTAGTGGGACACGGCT-3';

[0036] BIP: 5'-CGTGGTGTACGACGTGACGG-CGACGCACCTATCAATCTCG-3';

[0037] F3: 5'-CTGTAGAGCATGCTGCTGAT-3';

[0038] B3: 5'-CCCGCAGAGAAACCTATGG-3'.

[0039] Wherein, the forward inner primer FIP, the reverse inner primer BIP, the forward outer primer F3, and the reverse outer primer B3 can directly form a loop-mediated isothermal amplification detection primer composition for detecting Phytophthora syringae.

Embodiment 2

[0040] Example 2 Specificity test of Phytophthora syringa ring-mediated isothermal amplification reaction

[0041] In order to verify the specific primer sequence of Phytophthora syringae, the present embodiment uses 1 strain of Phytophthora syringae 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 syringae in diseased tissues. For moldy DNA, take 1 μL of DNA solution, add 23 μL of the detection solution prepared in Example 1 and 1 μL of sterilized deionized water to perform loop-mediated isothermal amplification reaction, and the reaction program is: 64°C for 60 min.

[0042] The specific method of extracting the DNA of each test material is as follows: take a small amount of mycelium powder, add 900 μL 2% CTAB extract and 90 μL 10% SDS, vortex and mix, place in a 55°C water bath for 1 hour, and invert several times every 10 minutes. Centrifuge at 12000rpm for 10min, take the super...

Embodiment 3

[0048] Example 3 Sensitivity test of Phytophthora syringa ring-mediated isothermal amplification reaction

[0049] In order to determine the sensitivity of the loop-mediated isothermal amplification detection method, the extracted DNA of Phytophthora syringae was measured with a spectrophotometer (1 μg / μL), 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 to carry out the loop-mediated isothermal amplification reaction, and the reaction program is: 64°C for 60 min. Take 2 μL of the amplification product and load the sample, the result is as follows figure 2 As shown, agarose gel electrophoresis and HNB chromogenic reaction are shown showing that the sensitivity of the loop-mediated isothermal amplification reaction reaches 100 pg of Phytophthora syringae DNA.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a LAMP detection primer combination of eugenopsis and a LAMP detecting kit and method thereof. The loop-mediated isothermal amplification detection primer combination is composed of a forward inner primer FIP, a backward inner primer BIP, a forward outer primer F3 and a backward outer primer B3. The detecting method is high in accuracy and specificity, convenient to operateand high in practicability, constant-temperature amplification is realized, a new technological platform is provided for detection of quarantine eugenopsis, the method can be used for high-sensitivity rapid detection of eugenopsis, pathogens are authenticated in the early stage of disease infection, and eugenopsis in import and export fruit samples can be detected. The method has important significance in reducing diseases caused by eugenopsis, reducing the blind use of pesticides, reducing production cost and reducing pesticide environment pollution.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a LAMP detection primer composition of Phytophthora syringae, a LAMP detection kit and a LAMP detection method thereof. Background technique [0002] Phytophthora ramorum belongs to Oomycota, Oomycetes, Peronosporales, Pythiaceae, Phytophthora. The host of the fungus has a wide range of distribution, mainly in the United States, Japan, Canada, the United Kingdom, France, Germany, Italy, Australia and other countries, and has not been reported in my country. The pathogen is mainly transmitted over long distances through infected young adult seedlings, soil and fruits. The pathogen mainly infects plants of 14 families and 29 genera including citrus fruits, Prunus fruits, pyracantha, clove, European chestnut, oak, etc., causing various lesions in roots, stems, leaves, and fruits. The extremely important plant pathogenic bacteria were included in the list of imported plant q...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6844C12Q2531/119
Inventor 戴婷婷焦彬彬郑香荣李中言王明远
Owner NANJING FORESTRY UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap