Multiple PCR detection kit for pathogens of respiratory tract, application and application method thereof

A technology for detection kits and respiratory tracts, applied in the field of nucleic acid amplification, can solve the problems of valuable information for patients, obstacles to large-scale use, and limited detection items, and achieve the effects of shortening performance, shortening detection time, and saving time

Active Publication Date: 2019-03-19
ZYBIO INC
View PDF5 Cites 10 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, several problems with traditional molecular diagnostic methods have hindered the large-scale use of this method in respiratory viral infections
Firstly, the detection process is relatively long, and the molecular diagnosis operation is complicated, involving nucleic acid extraction and product amplification. How to simplify the operation process and save experimental time is of great significance for patients to receive correct treatment quickly; secondly, most of the current molecular diagnostic tests are single-plex However, there are many types of respiratory viruses. There are about 145 serotypes of the most common rhinovirus that causes the common cold alone. The single-plex PCR system often has negative test results for patients, so it cannot give patients effective treatment. At present, there are many methods for detecting respiratory pathogens at home and abroad, each of which has its own advantages and disadvantages. The detection technologies include traditional isolation and cultur...

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
  • Multiple PCR detection kit for pathogens of respiratory tract, application and application method thereof
  • Multiple PCR detection kit for pathogens of respiratory tract, application and application method thereof
  • Multiple PCR detection kit for pathogens of respiratory tract, application and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0118] Determination of solution components in the nucleic acid extraction reagent of embodiment 2

[0119] 1. Reaction system

[0120] 1) PCR reaction solution

[0121] 10×Buffer for Taq 15%; MgCl 2 dATP 0.2 mM; dCTP 0.2 mM; dGTP 0.2 mM; dUTP 0.4 mM; BSA 1.5 mg / mL and glycerol 5%.

[0122] 2) PCR enzyme solution

[0123] Taq DNA Polymerase 15%; UNG 5%; Superscript V 4%; DTT 2mM; Glycerol 40%; Anti-Taq 15%.

[0124] 3) primer pair (same as in Example 1)

[0125] 4) Probe (same as in Example 1)

[0126] 5) Nucleic acid extraction product: using the nucleic acid extraction reagent of the present invention, 200 μL of clinical samples (nasopharyngeal swab or lung lavage fluid) were loaded, and 65 μL of elution buffer was eluted as the nucleic acid extraction product.

[0127] 2. Nucleic acid extraction reagents

[0128] Nucleic acid extraction reagent 1

[0129] Wherein the lysis binding solution includes the following components: 1% fatty acid methyl ester ethoxylate sod...

Embodiment 3

[0142] Example 3 Performance Verification of Detection Kit Primer Probes in Single and Multiplex PCR Systems

[0143] 1. Reaction system

[0144] 1) PCR reaction solution

[0145] 10×Buffer for Taq 15%; MgCl 2 dATP 0.2 mM; dCTP 0.2 mM; dGTP 0.2 mM; dUTP 0.4 mM; BSA 1.5 mg / mL and glycerol 5%.

[0146] 2) PCR enzyme solution

[0147] Taq DNA Polymerase 15%; UNG 5%; Superscript V 4%; DTT 2mM; Glycerol 40%; Anti-Taq 15%.

[0148] 3) primer pair: consistent with that in Example 1

[0149] 4) probe: consistent with that in Example 1

[0150] 5) Nucleic acid extraction product: consistent with that in Example 1

[0151] 2. Experimental process: select clinical samples of influenza A, influenza B, respiratory syncytial virus, parainfluenza 1, parainfluenza II, parainfluenza III, adenovirus, metapneumovirus, enterovirus or rhinovirus, for the detection of influenza A Influenza virus, the national reference product S1 is diluted E1, E2, E3, E4, E5, E6 times, the corresponding co...

Embodiment 4

[0157] Embodiment 4 detection kit specific detection

[0158] 1. Reaction system

[0159] 1) PCR reaction solution

[0160] 10×Buffer for Taq 15%; MgCl 2 dATP 0.2 mM; dCTP 0.2 mM; dGTP 0.2 mM; dUTP 0.4 mM; BSA 1.5 mg / mL and glycerol 5%.

[0161] 2) PCR enzyme solution

[0162] Taq DNA Polymerase 15%; UNG 5%; Superscript V 4%; DTT 2mM; Glycerol 40%; Anti-Taq 15%.

[0163] 3) primer pair: consistent with that in Example 1

[0164] 4) probe: consistent with that in Example 1

[0165] 5) Nucleic acid extraction product: consistent with that in Example 1

[0166] 2. Experimental process: Select clinical samples of other common respiratory pathogens, including Mycoplasma pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae and human genome, for nucleic acid extraction, and compare the experimental results of each item in the multiplex PCR system. In the process of nucleic acid extraction, add internal quality control plasmid amplicon sequence SEQ ID NO:31, and add in...

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 a multiple PCR detection kit for pathogens of respiratory tract, an application and an application method thereof. The PCR detection kit comprises a mixture of various primers and probes, a PCR reaction liquid, a PCR enzyme liquid, an inner quality control product and a nucleic acid extraction reagent. The detection kit is respiratory syncytial virus used for detecting one or more pathogens of influenza a virus, influenza B virus, respiratory syncytial virus, parainfluenza I, parainfluenza II, parainfluenza III, adenovirus, human metapneumovirus and enterovirus or rhinovirus. The application of the kit comprises the following steps: firstly, carrying out sampling; then carrying out PCR amplification and detection; and finally, obtaining a detection result. By combingand coordinating the nucleic acid extraction reagent, the primer pairs, the probes, a PCR program, the PCR reaction liquid and the PCR enzyme liquid, the technical effects of shortening the detectiontime and being good in detection sensitivity and good in specificity are achieved finally.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid amplification, and mainly relates to a kit for detecting respiratory pathogens, its application and its application method. Background technique [0002] Respiratory tract infection is very common in clinical practice, but due to the variety of pathogens that cause respiratory tract infection, rapid and accurate identification of pathogens is of great significance for clinical diagnosis and treatment and prevention of drug abuse. Fluorescent quantitative PCR technology based on Taqman probes can specifically amplify a large number of pathogenic nucleic acid molecules, and at the same time use the accumulation of fluorescent signals to realize the interpretation of negative and positive results. Compared with biochemical indicators and immune methods, molecular diagnosis is extremely sensitive, and can identify pathogen types and subtypes based on the differences in gene sequences of different...

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
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 陈威董璐欧阳许芬
Owner ZYBIO INC
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