Multi-liquid-phase gene chip detection primer, kit and method for quickly distinguishing 5 respiratory pathogens of mouse

A technology of liquid-phase gene chips and detection primers, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve difficult problems, achieve high accuracy, avoid cross-hybridization, and The effects of amplification and specificity

Active Publication Date: 2017-11-03
GUANGDONG LAB ANIMALS MONITORING INST
View PDF11 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with conventional PCR, fluorescent quantitative PCR has advantages in sensitivity, specificity, and speed. However, real-time fluorescent PCR technology is limited by the type of fluorescence and the instrument itself. It can only detect up to 5 targets, and the success of the experiment is extremely difficult. Big

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
  • Multi-liquid-phase gene chip detection primer, kit and method for quickly distinguishing 5 respiratory pathogens of mouse
  • Multi-liquid-phase gene chip detection primer, kit and method for quickly distinguishing 5 respiratory pathogens of mouse
  • Multi-liquid-phase gene chip detection primer, kit and method for quickly distinguishing 5 respiratory pathogens of mouse

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: A multiplex liquid-phase gene chip detection primer for rapidly distinguishing 5 kinds of mouse respiratory pathogens

[0058] After screening a large number of primers designed, it was found that the primer pairs A1 and A2, B1 and B2, C1 and C2, D1 and D2, E1 and E2 were paired for rapid detection of mouse pneumonia virus (PVM), hantavirus (HV ), Sendai virus (SV), lymphocytic choriomeningitis virus (LCMV), and mycoplasma pneumoniae (M.P.) have the best results. Confirmed by conventional electrophoretic detection. Its nucleotide sequence is as follows:

[0059] The nucleotide sequence of the primer pair that is used to detect mouse pneumonia virus (PVM) is as follows:

[0060] A1: 5'-AGATCACAGAGCCCGTCAAAAT-3' (SEQ ID NO: 1);

[0061] A2: 5'-GCATATAACATCCAATACGAGTTTGAA-3' (SEQ ID NO: 2);

[0062] The nucleotide sequence of the primer pair that is used to detect Hantavirus (HV) is as follows:

[0063] B1: 5'-GGACACAATCAATGGGGATACAAC-3' (SEQ ID NO: 3);

...

Embodiment 2

[0082] Example 2: A multiplex liquid-phase gene chip detection kit for rapidly distinguishing 5 kinds of mouse respiratory pathogens

[0083] The test kit includes the following components:

[0084] (1) multiple liquid-phase gene chip detection primers designed in embodiment 1;

[0085] (2) Five kinds of fluorescently encoded microspheres containing anti-tag sequences encoded with different fluorescent colors, and the anti-tag sequences can be complementary to the tag sequences in the detection primers of multiple liquid-phase gene chips; five kinds of microspheres All were purchased from luminex company, and the specific PVM, HV, LCMV, SV, and M.P. corresponding to the fluorescent coded microsphere numbers are MTAG-A015, MTAG-A065, MTAG-A042, MTAG-A062, and MTAG-A034;

[0086](3) streptavidin-phycoerythrin complex;

[0087] (4) Other reagents for PCR amplification reaction system.

[0088] The liquid-phase gene chip kit of the present invention includes the entire detectio...

Embodiment 3

[0089] Example 3: Establishment of multiple liquid-phase gene chip detection method for 5 kinds of mouse respiratory pathogens

[0090] 1. Single primer RT-PCR amplification specificity verification

[0091] (1) Positive template preparation: RNA / DNA was extracted from five pathogenic nucleic acids in the respiratory tract of mice, and subjected to RT reverse transcription to prepare positive cDNA-specific templates.

[0092] The RNA reverse transcription reaction system is as follows:

[0093]

[0094] (2) Single-plex PCR specificity verification: Use single-plex primers A1A2, B1B2, C1C2, D1D2, and E1E2 to perform PCR amplification on the five pathogenic cDNAs of PVM, SV, LCMV, HV, and M.P. to verify the specificity of the single-plex primers. sex.

[0095] The single-plex PCR reaction system is as follows:

[0096]

[0097]

[0098] see results figure 1 , figure 2 , image 3 . figure 1 , figure 2 , image 3 The results showed that each specific primer cou...

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 multi-liquid-phase gene chip detection primer, kit and method for quickly distinguishing 5 respiratory pathogens of a mouse. The multi-liquid-phase gene chip detection primer is easy to operate; a target amplified fragment is obtained through PCR (Polymerase Chain Reaction); an amplified product, fluorescence coded microspheres and streptavidin-phycoerythrin are hybridized; an MFI (Mean Fluorescence Intensity) value is read through a detector so as to distinguish different types of viruses. The method disclosed by the invention can detect a pneumonia virus, a hantaan virus, a sendai virus, a lymphocytic choriomeningitis virus and mycoplasma pulmonis of the mouse at the same time, and is high in specificity, high in sensitivity and high in repetitiveness. Compared with the conventional detection method, the method disclosed by the invention realizes simultaneous detection of various different target molecules in the same sample; the sample use amount is small; the operation is simple and quick; the detection cost can be greatly reduced.

Description

technical field [0001] The invention belongs to the field of detection of pathogenic microorganisms in experimental animals, and in particular relates to a method for rapidly distinguishing five kinds of mouse respiratory pathogens-mouse pneumonia virus (PVM), hantavirus (HV), Sendai virus (SV), lymphocyte choroid meninges Multiplex liquid-phase gene chip detection primers, kits and methods for inflammatory virus (LCMV) and mycoplasma pneumoniae (M.P.). Background technique [0002] Experimental animals are used as research materials, and their standardization is crucial, which is an important guarantee for the scientificity and repeatability of research data and conclusions. Microbiological quality testing of experimental animals is an important indicator for evaluating the quality of experimental animals. my country's national standard stipulates that the microorganisms that need to be excluded from mice without specific pathogens include 16 kinds of bacteria and 11 kinds ...

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): C12Q1/70C12Q1/68C12Q1/04C12N15/11C12R1/93C12R1/35
CPCC12Q1/6813C12Q1/686C12Q1/689C12Q1/701C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107C12Q2563/143C12Q2563/149
Inventor 王静郭鹏举黄韧张钰
Owner GUANGDONG LAB ANIMALS MONITORING INST
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