Multiplex real-time PCR kit and method for detecting respiratory pathogens and application

A kit and pathogen technology, applied in the field of biomedicine, can solve the problems of the narrow detection range of respiratory virus nucleic acid detection kits, the detection throughput is limited by the instrument, and the detection cycle is prolonged, so as to shorten the detection time, reduce the detection cost, and improve the detection efficiency. wide range of effects

Pending Publication Date: 2020-11-10
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although Mérieux’s FilmArray RP panel is easy to operate, fast and highly automated, it must be implemented with the help of the FilmArray automated analysis platform, and the scope of use is limited
Although Hirsch’s 13 kinds of respiratory pathogen multiple detection kits can realize multiple detection in one tube reaction, in addition to relying on the PCR machine, a special capillary electrophoresis instrument is required to analyze the products. Not only the capillary electrophoresis analysis process will prolong the detection period, And opening the cap of the amplified product is likely to cause laboratory aerosol pollution
Boao's six respiratory virus nucleic acid detection kits have a narrow detection range and must be implemented with the help of a supporting constant temperature amplification microfluidic nucleic acid analyzer. In addition, the detection throughput is limited by the instrument

Method used

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  • Multiplex real-time PCR kit and method for detecting respiratory pathogens and application
  • Multiplex real-time PCR kit and method for detecting respiratory pathogens and application
  • Multiplex real-time PCR kit and method for detecting respiratory pathogens and application

Examples

Experimental program
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Effect test

Embodiment 1

[0052] This embodiment provides a real-time PCR multiplex detection kit for detecting influenza A virus, influenza B virus, human rhinovirus and human metapneumovirus, including probes for detecting influenza A virus and specific Primer pairs for the selective amplification of influenza A virus target genes, probes for detection of influenza B virus and primer pairs for specific amplification of influenza B virus target genes, probes for detection of human rhinovirus and specificity Primer pair for amplifying human rhinovirus target gene, probe for detection of human metapneumovirus and primer pair for specific amplification of human metapneumovirus target gene, MgCl 2 , dNTPsMixture, AMV reverse transcriptase, RNase inhibitor, Taq DNA polymerase, plasmid mixture containing the target gene and nuclease-free water;

[0053] This embodiment provides a method for detecting bronchial lavage fluid using the above kit:

[0054] 1. Extract the nucleic acid of bronchial lavage fluid ...

Embodiment 2

[0070] This embodiment is the same as Example 1, except that the clinical samples, the primers and probe concentrations amplified by each pathogenic microorganism, the system component concentration of the amplification reaction, and the conditions of the amplification reaction are different from Example 1: this implementation The clinical sample taken for example is sputum, the primer concentration of each pathogenic microorganism amplification is 0.1 μ M, and the probe concentration is 50 nM; the MgCl in the reaction buffer described in the amplification reaction system 2 The working concentration of dNTPs Mixture is 2mM, the working concentration of dNTPs Mixture is 350μM, the working concentration of AMV reverse transcriptase in the enzyme mixture is 4U / μL, the working concentration of RNase inhibitor is 35U / μL, and the working concentration of TaqDNA polymerase 4U / μL, the amplification conditions are shown in the table below:

[0071]

[0072] In this example, the CT v...

Embodiment 3

[0074]This embodiment is the same as Example 1, except that the clinical samples, the primers and probe concentrations amplified by each pathogenic microorganism, the concentration of components in the amplification reaction system and the conditions of the amplification reaction are different from Example 1: this implementation The clinical sample that example takes is alveolar lavage fluid, and the primer concentration that each pathogenic microorganism is amplified is 1.0 μ M, and probe concentration is 250 nM; 2 The working concentration of dNTPs Mixture is 4mM, the working concentration of dNTPs Mixture is 450μM, the working concentration of AMV reverse transcriptase in the enzyme mixture is 6U / μL, the working concentration of RNase inhibitor is 45U / μL, the working concentration of Taq DNA polymerase The concentration is 6U / μL; the amplification conditions are shown in the table below:

[0075]

[0076] In this example, the CT value of human rhinovirus is ≤40, and the ...

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Abstract

The invention relates to a multiplex real-time PCR kit and method for detecting respiratory pathogens and an application. The kit comprises a reagent for detecting influenza A viruses, influenza B viruses, human rhinoviruses and human metapneumoviruses; the reagent comprises a probe for detecting the viruses, a primer pair for specifically amplifying corresponding virus target genes, a reaction buffer solution, an enzyme mixture, a positive control and a negative control; and a detection channel of the influenza A viruses is FAM, a detection channel of the influenza B viruses is VIC, a detection channel of the human rhinoviruses is Texas Red, and a detection channel of the human metapneumoviruses is Cy5. The kit disclosed by the invention can be used for simultaneously detecting the four kinds of viruses, simplifies the operation process, shortens the detection time, improves the detection flux and reduces the detection cost while having high sensitivity and specificity, and can be universally applied to various fluorescent quantitative PCR instruments.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a multiple real-time PCR kit, method and application for detecting respiratory pathogens. Background technique [0002] At present, the clinical diagnosis of respiratory pathogens mainly relies on the traditional isolation and culture method. Although the culture method is the gold standard for clinical pathogen diagnosis, it still has certain limitations, including: (1) It is difficult or impossible to culture a large number of pathogens in vitro; (2) The positive rate is low and the false negative rate is high (often as high as 50%); (3) The detection takes a long time: routine detection takes 1-4 days, and some slow-growing pathogens need 3-4 weeks to obtain the culture results; (4) The operation is cumbersome: the operator has high requirements, and the result repeatability is not good. [0003] In addition to the traditional isolation and culture method, most of the det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2600/166
Inventor 应斌武李为民宋兴勃柯博文陆小军陈豪吴涛
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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