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Kit for detecting respiratory viruses and application thereof

A respiratory tract and kit technology, applied in the field of respiratory virus detection kits, can solve the problems of false negatives, long virus time, complicated and expensive electron microscope detection methods, etc., and achieve the effect of accurate detection

Active Publication Date: 2015-11-04
CAPITALBIO CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many methods for detecting common respiratory viruses, but all of them have shortcomings. For example, electron microscope detection method is complicated and expensive, virus isolation and culture takes a long time and there are many false negatives, and usually loses clinical significance after the culture results are obtained. The method detects virus-specific antibodies but can only detect one virus at a time

Method used

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  • Kit for detecting respiratory viruses and application thereof
  • Kit for detecting respiratory viruses and application thereof
  • Kit for detecting respiratory viruses and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, preparation and use thereof for detecting the kit of respiratory tract virus

[0038] 1. Preparation of kits for detecting respiratory viruses

[0039] The kit for detecting respiratory virus provided by the present invention consists of the following:

[0040] 1. Constant temperature amplification buffer

[0041] The solvent of the constant temperature amplification buffer is water, and the solute and concentration are as follows: 200mM Tris-HCL (pH 8.0), 50mM DTT, 10mM dNTP, 10mM rNTP, 80mM MgCl 2 , 450mM KCl, 15% by volume DMSO, 1M sorbitol, 20mM tetramethylammonium chloride.

[0042] 2. Constant temperature amplification enzyme solution

[0043] The solvent of the constant temperature amplification enzyme solution is water, and the solute and concentration are as follows: AMV reverse transcriptase 1U / μl, T7 RNA polymerase 5U / μl, ribonuclease H 0.5U / μl, pyrophosphatase 0.5U / μl, RNase inhibitor Agent 5U / μl, BSA 0.5μg / μl.

[0044] 3. A 24-chamber dis...

Embodiment 2

[0058] Example 2. Sensitivity and specificity analysis of the kit for detecting respiratory viruses

[0059] 1. Preparation of reference RNA nucleic acid

[0060] 1. Construction of plasmids containing viral target genes

[0061] (1) Plasmid containing influenza A virus NP target gene

[0062] Insert segment 1-1541 of the influenza A virus NP target gene sequence (Genbank number Sequence ID of the influenza A virus NP target gene sequence: gb|KM366530.1|, Update Date: 2014-9-27) into the pUC19 vector (product of Tiange Biochemical Company) between the multiple cloning sites EcoRⅤ to obtain the recombinant plasmid pUC19-InfA.

[0063] (2) Plasmids containing influenza A virus H1 subtype target genes

[0064] Segment 1-1698 of the target gene sequence of influenza A virus H1 subtype (Genbank number Sequence ID of the target gene sequence of influenza A virus H1 subtype: gb|GQ475727.1|, Update Date: 2009-8-19) It was inserted into the pUC19 vector (Tiange Biochemical Co., Ltd...

Embodiment 3

[0126] Embodiment 3, the detection of actual clinical sample

[0127] 1. Types of clinical samples

[0128] The clinical samples used in this example came from the nasopharyngeal swab samples collected by Shenzhen No. 3 Hospital (based on the principle of voluntariness of the collectors), and the collected swabs were stored in 3 mL of normal saline, a total of 560 cases.

[0129] 2. Extraction of viral nucleic acid in clinical samples

[0130] The kit used for the extraction of viral nucleic acid from clinical samples is QIAamp Viral RNA Mini Kit (Qiagen), and the extraction is performed as follows:

[0131] (1) Take 140 μl of physiological saline for storing clinical sample swabs in step 1 into a 1.5ml centrifuge tube;

[0132] (2) Add 560 μl Buffer AVL containing Carrier RNA mixture (i.e. 5.6 μl Carrier RNA mixture + 560 μl Buffer AVL to the centrifuge tube, vortex slightly for 15 seconds;

[0133] (3) After the transient centrifugation is completed, place it at room temp...

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Abstract

The invention discloses a kit for detecting respiratory viruses and an application thereof. The kit comprises a primer pair group for detecting the respiratory viruses, wherein the primer pair group consists of 16 primer pairs, the sequences of which are shown in sequences 1 to 32 in a sequence table. The kit provided by the invention supports high throughput, can be used for rapidly and accurately detecting the infection of conventional respiratory viruses, and can clinically acquire a detection result of 16 virus indexes in one hour, which is not only faster than the real-time fluorescence quantitative PCR method generally used at present, but also has an important significance for rapidly assisting and instructing the treatment and medicine application. Meanwhile, the multi-index detection can also be used for investigating regional epidemiology and monitoring the epidemic situation so as to research the prevalence state of respiratory virus infection in China.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid amplification, and relates to a kit for detecting respiratory viruses and an application thereof. Background technique [0002] Respiratory tract infection is divided into upper respiratory tract infection and lower respiratory tract infection. The main pathogens include bacteria, viruses, mycoplasma, chlamydia, fungi, etc., among which viral infections are the main ones, especially more than 80% of upper respiratory tract infections are viral . The disease can come on in all seasons and at any age, and it can be transmitted by droplets containing virus, droplets, or contaminated utensils. Often when the body's resistance is lowered, such as being exposed to cold, fatigue, rain, etc., viruses or bacteria that already existed or invaded from the outside will grow and multiply rapidly, leading to infection. Adults usually recover within 5-7 days. [0003] Infection of respiratory pathogens in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6865C12Q1/70C12Q1/701C12Q1/702C12Q2531/143C12Q2537/143Y02A50/30
Inventor 张岩盖伟邢婉丽宋翠丹程京
Owner CAPITALBIO CORP
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