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Multiplex fluorescence PCR kit, method and application for detecting respiratory tract pathogens

A multiple fluorescence and kit technology, applied in the field of biomedicine, can solve the problems of narrow detection range of respiratory virus nucleic acid detection kits, limited detection throughput by instruments, limited use range, etc., so as to shorten the detection time and reduce the detection cost. , high sensitivity and specificity

Pending Publication Date: 2020-11-17
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
Hirsch’s Respiratory Pathogen Multiple Detection Kit can achieve multiple detections in one tube reaction, but in addition to relying on the PCR machine, it also needs a special capillary electrophoresis instrument to analyze the products. Not only the capillary electrophoresis analysis process will prolong the detection cycle, but also expand the Opening the lid of the product can also easily cause laboratory aerosol pollution
Boao's respiratory virus nucleic acid detection kit has 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, up to 16 channels

Method used

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  • Multiplex fluorescence PCR kit, method and application for detecting respiratory tract pathogens
  • Multiplex fluorescence PCR kit, method and application for detecting respiratory tract pathogens
  • Multiplex fluorescence PCR kit, method and application for detecting respiratory tract pathogens

Examples

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Effect test

Embodiment 1

[0051] This example provides a multiple real-time PCR detection kit for detecting adenovirus, cytomegalovirus, bocavirus and mycoplasma pneumoniae, including probes for detecting adenovirus and specific amplification of adenovirus target genes Primer pair for detection of cytomegalovirus and primer pair for specific amplification of cytomegalovirus target gene, probe for detection of Boca virus and primer pair for specific amplification of Boca virus target gene, Probe for detecting Mycoplasma pneumoniae and primer pair for specific amplification of Mycoplasma pneumoniae target gene, MgCl 2 , dNTPs Mixture, Taq DNA polymerase, plasmid mixture containing the target gene and nuclease-free water;

[0052] This embodiment provides a method for detecting bronchial lavage fluid using the above kit:

[0053] 1. Extract the nucleic acid of bronchial lavage fluid from clinical samples, and obtain the samples to be tested after purification;

[0054] 2. Prepare reaction system

[005...

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 TaqDNA polymerase is 2mM, the working concentration of dNTPs Mixture is 350μM, and the working concentration of TaqDNA polymerase is 4U / μL. The amplification conditions are shown in the following table:

[0071]

[0072] In this example, the CT value of cytomegalovirus was ≤40, the detection results of negative control, adenovirus, boca virus and mycoplasma pneumoniae had no C...

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 Taq DNA polymerase is 4mM, the working concentration of dNTPs Mixture is 450μM, and the working concentration of Taq DNA polymerase is 6U / μL; the amplification conditions are shown in the following table:

[0075]

[0076] In this example, the CT value of Boca virus was ≤40, the test results of negative control, adenovirus, Boca virus and Mycoplasma pneumoniae had no CT value, and the CT value of each channel detection target of the ...

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Abstract

The invention relates to a multiplex fluorescence PCR kit, a method and application for detecting respiratory pathogens. The kit comprises reagents for detecting adenoviruses, cytomegaloviruses, bocaviruses and mycoplasma pneumoniae, the reagents comprise probes for detecting the above viruses, primer pairs for specifically amplifying corresponding virus target genes, reaction buffer solutions, anenzyme mixture, a positive control and a negative control; and the detection channel of the adenovirus is FAM, the detection channel of the cytomegalovirus is VIC, the detection channel of the bocavirus is Texas Red, and the detection channel of the mycoplasma pneumoniae is Cy5. The kit disclosed by the invention can be used for simultaneously detecting the above four viruses, and has high sensitivity and specificity, at the same time, the operation process is simplified, the detection time is shortened, the detection flux is improved, the detection cost is reduces, and the kit 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 fluorescent 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: Such as viruses, anaerobic bacteria, fastidious bacteria, etc.; (2) low positive rate, high false negative rate (often up to 50%); (3) long detection time: routine detection takes 1-4 days, some slow growth Type pathogens need 3-4 weeks to obtain the culture results; (4) cumbersome operation: high requirements for operators and poor reproducibility of results. [0003] In addition to the traditional isolation and culture method,...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/35
CPCC12Q1/701C12Q1/689C12Q1/686C12Q2561/101C12Q2561/113C12Q2563/107C12Q2545/114C12Q2537/143
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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