Kit for detecting respiratory virus and its application

A kit and respiratory technology, applied in the field of kits for the detection of respiratory viruses, can solve the problems of time-consuming virus, complex and expensive electron microscopy detection methods, false negatives, etc.

Active Publication Date: 2017-10-27
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 virus and its application
  • Kit for detecting respiratory virus and its application
  • Kit for detecting respiratory virus and its application

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 constant temperature amplification buffer is water, 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, BSA0.5μg / μl.

[0044] 3. A 24-chamber disc chip loaded...

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 respiratory virus target genes

[0061] (1) Plasmids containing respiratory syncytial virus target genes

[0062] Insert the segment 5680-7390 of the RSV target gene sequence (Genbank number Sequence ID of the RSV target gene sequence: gb|KJ627648.1|, Update Date: 2014-4-16) into the pUC19 vector (day With the multiple cloning site EcoRⅤ of the biochemical company product), the recombinant plasmid pUC19-RSV was obtained.

[0063] (2) Plasmids containing rhinovirus target genes

[0064] Insert the segment 1-1175 of the rhinovirus target gene sequence (Genbank number Sequence ID: gb|M16248.1|, Update Date: 2003-2-7) of the rhinovirus target gene sequence into the pUC19 vector (product of Tiange Biochemical Company) ) between the multiple cloning sites EcoRⅤ to obtain the recom...

Embodiment 3

[0090] Embodiment 3, the detection of actual clinical sample

[0091] 1. Types of clinical samples

[0092] 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.

[0093] 2. Extraction of viral nucleic acid in clinical samples

[0094] 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:

[0095] (1) Take 140 μl of physiological saline for storing clinical sample swabs in step 1 into a 1.5ml centrifuge tube;

[0096] (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;

[0097] (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 virus and application thereof. The kit provided by the present invention contains a primer pair set for detecting respiratory viruses, consisting of 4 primer pairs whose sequence is sequence 1‑8 in the sequence listing. The kit provided by the invention supports high-throughput and can quickly and accurately detect common respiratory virus infections. For clinical purposes, the detection results of four virus indicators can be obtained within 1 hour, which is not only faster than the real-time fluorescence currently commonly used Quantitative PCR method is also of great significance for rapid auxiliary guidance of treatment and medication. At the same time, multi-indicator detection can also be used in regional epidemiological investigations and epidemic monitoring to study the prevalence of respiratory virus infections 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 Patents(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
Inventor 张岩盖伟邢婉丽马桂红程京
Owner CAPITALBIO CORP
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