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Primer combination and plasmid for simultaneously detecting 9 kinds of respiratory tract viruses and detection kit

A combination of primers and respiratory technology, applied in the direction of using vectors to introduce foreign genetic material, biochemical equipment and methods, microorganisms, etc., to achieve good social benefits, shorten the treatment cycle, and save medical expenses

Inactive Publication Date: 2021-02-05
GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technology has high sensitivity and specificity and can be applied to rapid diagnosis, but the detection requires the use of a fluorescence microscope or a related fluorescence detector, and can only detect respiratory viruses with corresponding antibodies

Method used

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  • Primer combination and plasmid for simultaneously detecting 9 kinds of respiratory tract viruses and detection kit
  • Primer combination and plasmid for simultaneously detecting 9 kinds of respiratory tract viruses and detection kit
  • Primer combination and plasmid for simultaneously detecting 9 kinds of respiratory tract viruses and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The design of embodiment 1 multiplex primer and the construction of positive control plasmid

[0026] 1. Design of multiple primers: for human internal reference GAPDH gene, influenza A virus M gene, influenza B virus HA gene, H7 influenza virus HA gene, NL63 coronavirus N gene, 229E coronavirus N gene, OC43 Bioinformatics analysis of type coronavirus N gene, type 1 parainfluenza virus HN gene, type 2 parainfluenza virus HN gene, boca virus NP1 gene, type A respiratory syncytial virus F gene and type B respiratory syncytial virus N gene . Through the public BLAST software in Genbank, the sequences of these genes were compared with other microorganisms, and sequence segments with higher specificity were selected. Then use the software Primer 5.0 to design a pair of upstream and downstream primers in this specific sequence, and add TAG sequence and C18 connection sequence to the upstream primer respectively, and add biotin labeling sequence to the downstream primer. The...

Embodiment 2

[0036] Example 2 Selection of Multiplex Amplification Primers—Evaluation of Primer Specificity

[0037] According to the primers and plasmids of Example 1, 12 kinds of primers are mixed for detection, and other 11 kinds of templates that lack the corresponding primer amplification templates that need to be verified are mixed as detection templates for PCR amplification, and the corresponding primers that need to be verified are mixed. The amplified template was used as a positive control, and water was used as a negative control to evaluate the specificity of the corresponding primers.

[0038] Specific steps are as follows:

[0039] (1) Preparation of PCR system (on ice, 0-4°C, one sample volume)

[0040] 2×PCR Reaction Mix 10μl, Primer 4.8μl, nucleic acid sample 2μl, total volume 20μl.

[0041] (2) Set the PCR program

[0042] Maintain at 95°C for 30 sec; maintain at 95°C for 5 sec, and maintain at 60°C for 30 sec, read the plate, and repeat 40 cycles; analyze the melting...

Embodiment 3

[0044] Example 3 Evaluation of the detection sensitivity of the independent amplification of 10 kinds of primer amplification systems

[0045] 1. DNA detection sensitivity evaluation

[0046] Specific steps are as follows:

[0047] (1) Preparation of PCR system (on ice, 0-4°C, one sample volume)

[0048] 2×PCR Reaction Mix 10μl, Primer 0.8μl, nucleic acid sample 2μl, total volume 20μl.

[0049] (2) Set the PCR program

[0050] Keep at 95°C for 3min; keep at 95°C for 30sec, keep at 58°C for 30sec, read the plate, keep at 72°C for 30sec, repeat 40 cycles; analyze the melting curve; store at 4°C.

[0051] (3) Preparation for detection target

[0052] The positive control plasmids of the 10 amplification systems were diluted 10-fold, and added to the detection system, using water as a negative control, for PCR amplification and fluorescence detection, and the detection sensitivity curve was drawn. The results are as follows: Figure 13-22 As shown, the results of 3 repeated e...

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Abstract

The invention discloses a primer combination and plasmid for simultaneously detecting 9 kinds of respiratory tract viruses and a detection kit and belongs to the technical field of biology. The primercombination comprises primer pairs for detecting an influenza a virus, an influenza b virus, an H7 influenza virus, an NL63 corona virus, a 229E corona virus, a type 2 parainfluenza virus, Bocavirus,a respiratory syncytial virus A, a respiratory syncytial virus B and a human internal-reference GAPDH gene. The detection kit comprises a 2*multiple reverse transcription PCR reaction solution, a reverse transcription PCR enzyme mixed solution, a primer mixed solution, non-RNAase water, a magnetic bead mixed solution, a reported buffer solution, a positive control and phycoerythrin-modified streptavidin. According to the primer combination and plasmid and the detection kit, by virtue of a liquid-phase chip technology, the manufactured detection kit can be used for carrying out qualitative analysis on a variety of respiratory tract viruses simultaneously, and the sensitivity and specificity of detection reach PCR levels; and types of respiratory tract virus infections can be rapidly and accurately judged, early etiologic diagnosis and treatment on clinical respiratory tract infections are facilitated, the shortening of a treatment cycle is facilitated, the medical expense is reduced, and good social benefits are generated.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer combination, a plasmid and a detection kit capable of simultaneously detecting nine kinds of respiratory viruses. Background technique [0002] Respiratory viral infections are one of the leading causes of human morbidity and mortality worldwide. So far, most acute upper and lower respiratory diseases are caused by pathogens other than bacteria, among which respiratory viruses are the most common. Respiratory viral infection can cause multiple serious complications, the greatest harm of which is its potential to cause a region, a country or even a worldwide pandemic. Therefore, respiratory virus infection has become a serious global public health problem. [0003] At present, the traditional laboratory diagnostic methods for respiratory virus infection in various medical institutions at home and abroad mainly include virus isolation, virus antigen and antibody analysis, an...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12N15/63C12R1/93
CPCC12Q1/701C12Q1/686C12N15/63C12Q2600/166C12Q2600/16C12Q2537/143C12Q2545/113C12Q2545/101C12Q2521/107
Inventor 龙飞黄庆珊周焕杰梁振宇王徐飞肖静彭涛陈荣昌
Owner GUANGZHOU MEDICAL UNIV
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