Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Primer combination for detecting 2 soft rot pathogens of orchids, and detection method

A combination of primers and pathogens technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/testing, etc., can solve problems such as undiscovered

Pending Publication Date: 2020-07-07
NINGDE NORMAL UNIV
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since oncidium, dendrobium and phalaenopsis are currently planted on a large scale, no germplasm resistant to soft rot has been found.

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
  • Primer combination for detecting 2 soft rot pathogens of orchids, and detection method
  • Primer combination for detecting 2 soft rot pathogens of orchids, and detection method
  • Primer combination for detecting 2 soft rot pathogens of orchids, and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] like figure 1 As shown, the leaves of Phalaenopsis orchids were inoculated with Erwinia carotovora subsp. Dickia oncidium leaves; healthy Dendrobium leaves as materials.

[0042] DNA extraction:

[0043] Cut 0.5 g of healthy leaves and leaves at the junction of disease and health, respectively, and use CTAB method to extract DNA.

[0044] Primer synthesis:

[0045] Synthesize pathogen-specific primers and Orchidaceae internal reference primers according to Table 1. Primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.

[0046] The PCR reaction system is 20 μL: 1 μL of DNA template, 1 μL of 10 μmol / L different upstream and downstream primers, 10 μL of 2×TaqPCR Mix reaction solution, add dd H 2 O to a total volume of 20 μL.

[0047]The PCR reaction system was 20 μL; the PCR reaction conditions were: pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 20 s, annealing at 52-56 °C for 20 s, extension at 72 °C for 30 s, a total of 40 cycl...

Embodiment 2

[0060] Oncidium leaves inoculated with Dikkerella for 24 h

[0061] DNA extraction:

[0062] Cut 0.5 g of leaves at the junction of diseased and healthy leaves, homogenate with 1 mL of sterile water, centrifuge to get the supernatant, and use sterile water to dilute to 1, 10, 100, 1000, 10000, 100000 times dilutions respectively for later use.

[0063] The PCR reaction system is 20 μL: 1 μL of the above diluent template, 10 μmol / L of 1 μL of each of the 3 primers for D. dactyla, 10 μL of 2×Taq PCR Mix reaction solution, add dd H 2 O to a total volume of 20 μL.

[0064] The PCR reaction system was 20 μL; the PCR reaction conditions were: pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 20 s, annealing at 52-56 °C for 20 s, extension at 72 °C for 30 s, a total of 40 cycles; extension at 72 °C for 6 min.

[0065] 1% agarose electrophoresis to detect 1-6 μL amplification products, such as image 3 As shown, it can be seen that this detection method can detect orc...

Embodiment 3

[0067] Cut and inoculate 0.5 g of leaves at the junction of diseased and healthy leaves with Diektolia and Erwinia carotovora subsp. Figure 4 ), 1mL sterile water homogenate, centrifuge to take the supernatant, and use sterile water to dilute to 1, 10, 100, 1000, 10000, 100000 times dilution respectively for later use.

[0068] The PCR reaction system is 20 μL: 1 μL of the above diluent template, 1 μL of 10 μmol / L different upstream and downstream primers, 10 μL of 2×Taq PCR Mix reaction solution, add dd H 2 O to a total volume of 20 μL.

[0069] The PCR reaction system was 20 μL; the PCR reaction conditions were: pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 20 s, annealing at 52-56 °C for 20 s, extension at 72 °C for 30 s, a total of 40 cycles; extension at 72 °C for 6 min.

[0070] Figure 5 Among them, the primer combination of different lane reaction tubes is as follows

[0071] Lane 1: Dickeya primers Dickeya F85 and Dickeya R480;

[0072] Lane 2: ...

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

PropertyMeasurementUnit
Sensitivityaaaaaaaaaa
Login to View More

Abstract

The invention provides a primer combination for detecting 2 soft rot pathogens of orchids, and a detection method. Six primers are designed with a common specific fragment of the two pathogens and canbe used for detection of an Erwinia carotovora subsp.carotovora pathogen and a Dickeyadadantii pathogen of the orchids. Six primers are designed aiming at three common characteristic loci of the twopathogens, so that two positive segments can be generated by a true positive sample and used for elimination of false positivity; and design for eliminating false genitive primers is added, wherein one positive segment can be generated by true negativity through design of loci for two common characteristic loci aiming at oncidium, dendrobium and phalaenopsis. The primer combination and the detection method have low requirements for equipment and a whole course can be completed within only 2 h at the soonest.

Description

technical field [0001] The invention relates to the field of plant virus detection, in particular to a primer combination and a detection method for detecting two kinds of soft rot pathogens of Orchidaceae plants. Background technique [0002] Soft rot is an important disease of a variety of cultivated crops. Orchidaceous plants are very susceptible to soft rot. Erwinia carotovora subsp. Erwinia carotovora subsp. carotovora ) and Diektia dactica ( Dickeya dadantii ) is the main pathogen causing orchid soft rot, once in oncidium (Oncidium) , Dendrobium ( Dendrobium ),Phalaenopsis( Phaleanopsis ) and other soft rot diseased plants were found. The two have a close relationship, and both rely on secreted pectinase, polygalacturonase and other extracellular enzymes to degrade the cell wall of the host plant to cause pathogenicity. They are highly contagious and destructive. It can lead to large-scale disease of orchid plants, causing a large number of plants to die. ...

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): C12Q1/689C12Q1/04C12N15/11C12R1/18C12R1/01
CPCC12Q1/689C12Q2600/166
Inventor 郑世仲叶炜王培育江胜滔陈美霞
Owner NINGDE NORMAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com