RPA primers, probe sets and kit for detecting respiratory viruses

A virus detection and respiratory technology, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of restricting the popularization and application of RPA technology, the difficulty of RPA primers, and the lack of good software analysis of RPA primers. Achieve good specificity, high sensitivity, and good specificity

Pending Publication Date: 2019-11-26
中国人民解放军疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, due to the lack of good software analysis for the design and screening of RPA primers, it is relatively difficul

Method used

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  • RPA primers, probe sets and kit for detecting respiratory viruses
  • RPA primers, probe sets and kit for detecting respiratory viruses
  • RPA primers, probe sets and kit for detecting respiratory viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Preparation of RPA primers and probe sets for respiratory virus detection

[0036] 1. Alignment of conserved sequences and construction of cloning vectors

[0037] (1) Download the cDNA or DNA sequences encoding the same protein of the three viruses from the NCBI gene bank (download at least 40 sequencing sequences from different regions in the past 5 years for each virus);

[0038] (2) Use DNAman software to compare sequences, find out relatively conserved sequences, and design and optimize primers and probes.

[0039] The determined conserved sequence is as follows:

[0040]

[0041]

[0042] 2. Design of primers and probes

[0043] RPA primer design method:

[0044] (1) Select 32-35 bases from the single strand of the conserved sequence of the virus compared as the upstream and downstream primers.

[0045] (2) Control the base length between the two primers between 200-400bp.

[0046] (3) The dNTP at the 5' end avoids guanine.

[0047] (4) Control the GC ...

Embodiment 2

[0069] Composition of kits for detection of respiratory viruses

[0070] Including primers, probe sets and detection reagents in Example 1.

[0071] The detection reagent preferably includes RPA buffer, dNTP, recombinase, polymerase, magnesium acetate and water, wherein the RPA buffer, dNTP, recombinase and polymerase are derived from the RPA-exo kit of Twist Company.

[0072] The method of use of the test kit:

[0073] (1) Dissolve the lyophilized tube containing dNTP, recombinant enzyme and polymerase dry powder with RPA buffer.

[0074] (2) Add upstream primers, downstream primers, templates and water for RPA amplification, and the total volume of each sample is 47.5 μL. The loading reagents and volumes are shown in Table 1.

[0075] (3) Amplification starts after adding 2.5 μL of magnesium acetate solution, shake and briefly centrifuge.

[0076] (4) Add the centrifuged solution system into a constant temperature fluorescence detector (GEN3-02-P1), set the temperature a...

Embodiment 3

[0098] Comparison between the detection results of the primers, probe sets and kits provided by the invention for respiratory viruses and the detection results of the fluorescent quantitative PCR method.

[0099] Taking adenovirus ADV as an example, the same sample was detected by fluorescent quantitative PCR and the kit method provided by the present invention, and the results were compared.

[0100] Real-time quantitative PCR detection of adenovirus ADV:

[0101] The upstream primer for fluorescent quantitative PCR detection: gaggagccagatattgatatggaatt (SE Q ID No.13);

[0102] Downstream primer: aattgacattttccgtgtaaagca (SEQ ID No.14);

[0103] Fluorescent probe: FAM-aagctgctgacgctttttcgcctga-BHQ1 (SEQ ID No. 15) for detection.

[0104] For the steps of the method for detecting adenovirus ADV with the kit provided by the present invention, refer to Example 2.

[0105] As can be seen from the results, through the continuous increase of the number of cycles, the detection ...

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Abstract

The present invention provides RPA primers, probe sets and a kit for detecting respiratory viruses, and belongs to the technical field of virus detection. The respiratory viruses include respiratory syncytial virus type A, coronavirus NL63 and adenovirus-7. Sequences of an upstream primer, a downstream primer and a fluorescent probe for detecting the respiratory syncytial virus type A are shown inSEQ ID No.1-3; sequences of an upstream primer, a downstream primer and a fluorescent probe for detecting the coronavirus NL63 are shown in SEQ ID No.4-6; and sequences of an upstream primer, a downstream primer and a fluorescent probe for detecting the adenovirus-7 are shown in SEQ ID No.7-9. The primers and probe sets have advantages of good specificity and high sensitivity, and can be used toquickly detect the respiratory syncytial virus type A, coronavirus NL63 and adenovirus type 7 on sites.

Description

technical field [0001] The invention belongs to the technical field of virus detection, in particular to RPA primers, probe sets and kits for respiratory virus detection. Background technique [0002] Respiratory tract infection is one of the most important causes of human morbidity and death. There are many types of pathogens that cause respiratory infections. 95% of acute upper respiratory diseases and most lower respiratory diseases are caused by pathogens other than bacteria, among which respiratory viruses are the most common, including respiratory syncytial virus, parainfluenza virus, coronavirus, nasal virus, human metapneumovirus, human bocavirus, etc. Because the clinical symptoms and signs of respiratory tract infections are very similar, the treatment methods for infections caused by different types of pathogens are quite different. Therefore, rapid and sensitive detection and identification of pathogenic microorganisms is helpful for clinical diagnosis and trea...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844Y02A50/30
Inventor 宋宏彬郝荣章郭旭东戚红卷赵荣涛张玉玺杨益张亚萍吴枫
Owner 中国人民解放军疾病预防控制中心
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