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

LAMP (loop-mediated isothermal amplification) double-strand detection probe and freeze-dried microsphere for burkholderia gladioli as well as preparation method and detection method of freeze-dried microsphere

A Holderella and detection probe technology, applied in the field of LAMP double-stranded detection probes for Burkholderia gladiolus, can solve the problem of low specificity and false positives, aerosol pollution, strong subjective factors, etc. problem, to achieve the effect of high accuracy, avoiding pollution, and not easy to false positive

Pending Publication Date: 2022-07-05
河南冠宇仪器有限公司 +1
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are mainly 1 methods for interpreting the results of the ring-mediated constant temperature amplification method: turbidity method, which can be used to observe whether there is white flocculent precipitate with the naked eye after the reaction, and determine the test result. The subjective factors are strong and the sensitivity is low.
2. Chromogenic method, add nucleic acid dyes such as SYBR GREEN, HNB, calcein, etc. after the reaction, and display different colors under ultraviolet light or naked eyes. The result is easy to judge, but it needs to be opened after the reaction, which is very easy to cause aerosol Pollution
3. Fluorescence detection method. By adding fluorescent dye SYBR GREEN to the reaction system, the fluorescence changes in the system can be observed in real time through a fluorescent PCR instrument to generate a fluorescence curve, and the amplification results can be observed through the curve. This method is closed-tube detection to eliminate gas Sol pollution, but due to the characteristics of SYBR GREEN dye, it can combine with any double-stranded DNA and generate fluorescence, which is low in specificity and prone to false positives

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 (loop-mediated isothermal amplification) double-strand detection probe and freeze-dried microsphere for burkholderia gladioli as well as preparation method and detection method of freeze-dried microsphere
  • LAMP (loop-mediated isothermal amplification) double-strand detection probe and freeze-dried microsphere for burkholderia gladioli as well as preparation method and detection method of freeze-dried microsphere
  • LAMP (loop-mediated isothermal amplification) double-strand detection probe and freeze-dried microsphere for burkholderia gladioli as well as preparation method and detection method of freeze-dried microsphere

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 LAMP double-stranded detection probe and primer design

[0046]The conserved sequences of Burkholderia gladiolus were screened out through literature review and comparison, and the primers and probes of the target sequences were designed using special LAMP primer design software, and the designed primers and probes were designed on Primer-blast. Analysis and optimization, primer and probe design principles and positions such as figure 1 As shown, the finalized probe and primer sequences are as follows:

[0047] The probe consists of two parts, one is a luminescent probe and the other is a quenching probe. The 5' end of the luminescent probe is labeled with a fluorescent light-emitting group, such as FAM, VIC, TET, etc. The 3' end of the quenching probe is labeled with Quenching light groups such as TAMRA, QSY, BHQ, etc., the luminescent probe is located on FIP, the fluorophore is labeled at the 5' end of FIP, the quenching probe is complementary to the F1C se...

Embodiment 2

[0053] The present embodiment provides a freeze-dried microsphere for detecting Burkholderia gladiolus, and its preparation method includes:

[0054] (1) Preparation of lyoprotectant: 70% trehalose, 8% glycine, and 25% PEG20000 were mixed in a volume ratio of 2:3:6 to obtain a lyoprotectant.

[0055] (2) Construct an amplification reaction system:

[0056] The total volume of the LAMP detection and amplification system constructed in this example is 15 μL, and the detection primer set shown in Table 1 is mixed with the LAMP reaction base liquid shown in Table 1 to obtain an amplification reaction system.

[0057] Table 1. Amplification reaction system

[0058] component name Concentration (μM) Addition amount (μL) Outer primer F3 / B3 100μM 0.05-0.1 Internal primer FIP / BIP 100μM 0.4-0.5 double-stranded detection probe 100μM 0.1-0.2 Bst DNA polymerase 8U 1-1.5 dNTPs 10mM each 3.0-4.0 betaine 5M 3-5 Reagent 200mM ...

Embodiment 3

[0061] Example 3 Establishment of LAMP detection reaction system

[0062] The total volume of the LAMP detection reaction system constructed in this example is 25 μL, which specifically includes:

[0063] The freeze-dried microspheres prepared in Example 2 were added to 26 μL of water for reconstitution, and the template DNA (2 μL) extracted from the sample to be tested was added to the reconstituted reaction system, and the reaction was carried out at 60 to 65 ° C for 50 to 70 min, LAMP amplification was performed; in this example, the amplification was performed at 63° C. for 60 min.

[0064] Result judgment:

[0065] (1) Visual method, directly observe the color change, so as to determine the LAMP amplification and the presence or absence of Burkholderia gladiolus in the sample.

[0066] (2) Fluorescence curve method, the fluorescence quantitative PCR instrument is used to collect the fluorescence of the luminescent group, and the amplification curve is given to judge the...

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 provides an LAMP (loop-mediated isothermal amplification) double-strand detection probe and freeze-dried microspheres of burkholderia gladioli as well as a preparation method and a detection method of the freeze-dried microspheres, and belongs to the field of gene detection. The double-chain detection probe comprises a light-emitting probe and a quenching probe, the light-emitting probe is located on the FIP, the 5'end of the FIP sequence is marked with a fluorescent light-emitting group, the quenching probe is complementary with the F1C sequence, and the 3 'end of the quenching probe is marked with a fluorescent quenching group. The LAMP detection freeze-dried microsphere comprises the double-stranded detection probe, the freeze-dried microsphere and the detection method, the burkholderia gladioli 16-23sRNA is taken as a detection target, a double-stranded probe-constant temperature color-changing system is adopted, a result can be interpreted by combining fluorescent quantitative PCR (Polymerase Chain Reaction) atlas analysis and a naked eye observation method, the probe is high in specificity, the color-changing color contrast is obvious, misjudgment is not easy to generate, and the detection result is accurate. The dual systems reflect each other, and the detection accuracy is high.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a LAMP double-stranded detection probe of Burkholderia gladiolus, freeze-dried microspheres and a preparation method and detection method thereof. Background technique [0002] Burkholderia gladiolus is a gram-negative brevis bacilli with blunt ends, no dentate spores, and flagella, which are widely distributed in nature. Burkholderia gladiolus is facultatively anaerobic and easily grows on food surfaces. Burkholderia gladiolus can produce rice yeast acid and cause food poisoning. Burkholderia gladiolus is not resistant to high temperature and is easily killed by high temperature, but the "rice yeast acid" it produces The toxin is heat-resistant and cannot be destroyed by ordinary cooking methods. [0003] At present, the commonly used detection methods for Burkholderia gladiolus include chromogenic medium culture method, fluorescence quantitative PCR, LAMP c...

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): C12Q1/689C12Q1/6844C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6844C12Q2531/119
Inventor 许世伟许远邵俊影吴镇宇王欣林圣博
Owner 河南冠宇仪器有限公司
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