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Kit for detecting enterovirus and application of kit

An enterovirus and kit technology, applied in the field of nucleic acid amplification, can solve the problems of many false negatives, loss of clinical significance, complicated and expensive electron microscopy detection methods, etc., and achieve the effect of accurate detection

Active Publication Date: 2015-12-02
CAPITALBIO CORP +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are many methods for detecting common enteroviruses, but all of them have deficiencies, such as 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. Immunoassays detect virus-specific antibodies but can only detect one virus at a time

Method used

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  • Kit for detecting enterovirus and application of kit
  • Kit for detecting enterovirus and application of kit
  • Kit for detecting enterovirus and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038]Embodiment 1, preparation and use thereof for detecting the kit of enterovirus

[0039] 1. Preparation of kits for detecting enteroviruses

[0040] The kit for detecting enterovirus provided by the present invention consists of the following:

[0041] 1. Constant temperature amplification buffer

[0042] The solvent of the constant temperature amplification buffer is water, and the solute and concentration are as follows: 200mM Tris-HCL (pH8.0), 50mM DTT, 10mMdNTP, 10mMrNTP, 80mMMgCl 2 , 450mM KCl, 15% by volume DMSO, 1M sorbitol, 20mM tetramethylammonium chloride.

[0043] 2. Constant temperature amplification enzyme solution

[0044] 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 H0.5U / μl, pyrophosphatase 0.5U / μl, RNase inhibition Agent 5U / μl, BSA0.5μg / μl.

[0045] 3. A 24-chamber disc chip loaded with primer pair...

Embodiment 2

[0059] Example 2. Sensitivity and specificity analysis of the kit for detecting enteroviruses

[0060] 1. Preparation of reference RNA nucleic acid

[0061] 1. Construction of plasmids containing enterovirus target genes

[0062] (1) Plasmids containing enterovirus target genes

[0063] Insert the 5-1705 segment of the enterovirus Entv target gene sequence (Genbank number SequenceID: gb|KC755230.1|, UpdateDate: 2013-12-17) of the enterovirus Entv target gene sequence into the pUC19 vector (Tiange Biochemical company product) between the multiple cloning site EcoRⅤ to obtain the recombinant plasmid pUC19-Entv.

[0064] (2) Plasmid containing enterovirus EV71 target gene

[0065] Insert the segment 4-1695 of the enterovirus EV71 target gene sequence (Genbank number SequenceID: gb|HM245928.1|, UpdateDate: 2011-9-27) of the enterovirus EV71 target gene sequence into the pUC19 vector (Tiange Biochemical company product) between the multiple cloning site EcoRⅤ to obtain the reco...

Embodiment 3

[0088] Embodiment 3, the detection of actual clinical sample

[0089] 1. Types of clinical samples

[0090] 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.

[0091] 2. Extraction of viral nucleic acid in clinical samples

[0092] The kit used for the extraction of viral nucleic acid from clinical samples is QIAampViralRNAMiniKit (Qiagen), and the extraction is performed as follows:

[0093] (1) Take 140 μl of physiological saline for storing clinical sample swabs in step 1 into a 1.5ml centrifuge tube;

[0094] (2) Add 560 μl BufferAVL containing CarrierRNA mixture (i.e. 5.6 μl CarrierRNA mixture + 560 μl BufferAVL to the centrifuge tube, vortex slightly for 15 seconds;

[0095] (3) After the transient centrifugation is completed, place it at room temperature ...

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Abstract

The invention discloses a kit for detecting enterovirus and application of the kit. The kit comprises a primer pair group for detecting the enterovirus; the primer pair group consists of three primer pairs; and sequences of the primer pairs are sequences 1-6 in a sequence list. The kit supports high through-put, and can detect enterovirus infection quickly and accurately; when the kit is used clinically, detection results of three kinds of enterovirus indexes can be obtained within 1 hour, the speed is higher than the speed in the existing frequently used real-time fluorescence quantitative PCR (polymerase chain reaction) method, and the kit has important significance in quick assistant guide treatment and pharmacy. Moreover, multi-index detection can also be used for regional epidemiological research and epidemic surveillance so as to research epidemic conditions of enterovirus 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 enteroviruses and an application thereof. Background technique [0002] Enteroviruses include poliovirus, Coxsackievirus (Coxsackievirus), enterocytopathic human orphan virus (enterocytopathichumanorphanvirus ECHO referred to as Echo virus) and a new type of enterovirus, a total of 71 serotypes. Enteroviruses are infectious diseases caused by viruses. The mild clinical manifestations are fatigue, fatigue, and low-grade fever. In severe cases, systemic infection can damage important organs such as the brain, spinal cord, heart, and liver. The prognosis is poor, and sequelae or cause death. This type of disease is distributed all over the world. It occurs all year round in the tropics and subtropics, and is more common in the temperate zone in summer. The incidence is high in warm, humid, poor sanitation and crowded areas. [0003] Hand-foot-m...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/70
Inventor 张岩盖伟邢婉丽宋翠丹程京周伯平单万水刘厚明
Owner CAPITALBIO CORP
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