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Kit for performing direct immunofluorescent detection on enterovirus 71 and coxsackievirus A16 and application thereof

A technology for immunofluorescence detection and coxsackie virus, which is applied in the field of pathogen detection, can solve the problems of easy pollution, long time-consuming, low sensitivity, etc., and achieve the effect of high-efficiency detection

Inactive Publication Date: 2017-09-01
GUANGDONG HECIN SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the equipment is expensive, the requirements for the experimental site are high, the requirements for the operator are high, it is easy to pollute, and it is easy to produce false positives and lead to misdiagnosis
[0009] (2) Diagnosis by comprehensive evaluation of clinical symptoms: this method cannot be diagnosed accurately in the early stage of the disease due to atypical symptoms
IgG antibody needs to be produced at least 14 days after the onset of illness, and generally a second infection may be detected in sufficient amount, therefore, this method cannot be used for early diagnosis
IgM antibody is produced 5-7 days after virus infection, and is limited by serum IgM concentration, resulting in low sensitivity and prone to false negatives
During the window period from virus infection to IgM antibody production, it is not applicable to use serum immunity to detect virus antibodies, so the method of detecting serum IgM is also very limited
[0011] The above three methods have disadvantages such as inconvenient operation, long time-consuming, and inaccurate diagnosis during clinical use.

Method used

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  • Kit for performing direct immunofluorescent detection on enterovirus 71 and coxsackievirus A16 and application thereof
  • Kit for performing direct immunofluorescent detection on enterovirus 71 and coxsackievirus A16 and application thereof
  • Kit for performing direct immunofluorescent detection on enterovirus 71 and coxsackievirus A16 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: the preparation of kit

[0044] 1 Cell collection and processing

[0045] 1.1 Cell collection: The kit of the present invention is used to detect EV71 and CA16 virus antigens in the sample cells, and EV71 and CA16 virus infection involves a wide range, therefore, theoretically, any cell sample that can be prepared into a single cell suspension is applicable In the kit of the present invention. Judging from the path of patients infected with EV71 and CA16 viruses and the experience of collecting samples, the collection of nasopharyngeal swabs is easy, non-invasive and fast, and the collection of patients after rinsing is basically free of impurities and does not need to be washed repeatedly. Therefore, the collection of cell samples is the first choice For nasopharyngeal swabs, the embodiments all take nasopharyngeal swabs as an example.

[0046] 1.2 Preparation of single-cell suspension: Put the test sample into a sampling tube containing 3ml PBS or viru...

Embodiment 2

[0057] Embodiment 2: Enterovirus 71 type and Coxsackie virus A16 type detection kit (flow cytometry)

[0058] This embodiment provides a kit for directly detecting Enterovirus 71 and Coxsackievirus A16 by using a flow cytometer. The kit can be used to assist clinical diagnosis of hand, foot and mouth disease.

[0059] 1. The components of the kit:

[0060]

[0061] Note: For the convenience of use, the antibody, preservative, antibody stabilizer and 1% Triton-X100 in the kit were all prepared as staining solution according to the operation method in Example 1, and packed in a brown bottle; is the mass volume ratio (g / 100ml);

[0062] 2. Preparation of reagents in the kit

[0063] 2.1 Composition of PBS and PBS-T

[0064]

[0065]

[0066] with dH 2 Dilute the volume of O to 1L to be the PBS solution, and add 0.05% tween-20 to the PBST solution on this basis;

[0067] 2.2 Cell fixation solution: 4% paraformaldehyde: 4g paraformaldehyde was dissolved in 100ml PBS ...

Embodiment 3

[0084] Embodiment 3: Enterovirus 71 type and Coxsackie virus A16 type detection kit (direct immunofluorescence method)

[0085] This embodiment provides a kit for directly detecting Enterovirus 71 and Coxsackievirus A16 using a fluorescence microscope. The kit can be used to assist clinical diagnosis of hand, foot and mouth disease.

[0086] 1 The kit consists of:

[0087]

[0088]

[0089] Quality Control Slides

[0090] Note: For the convenience of use, the antibodies, preservatives, antibody stabilizers, Evans Blue and 1% Triton-X100 in the kit were all prepared as fluorescent identification reagents according to the operation method in Example 1, and packed in a brown bottle Medium; Percentages are mass volume ratio (g / 100ml);

[0091] 2 Preparation of reagents in the kit

[0092] 2.1 PBS

[0093]

[0094] with dH 2 O dilute to 1L of PBS solution.

[0095] 2.2 Cell fixation solution: Prepare by dissolving 4g of paraformaldehyde in 100ml of PBS solution.

...

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Abstract

The invention provides a kit for performing direct immunofluorescent detection on enterovirus 71 and coxsackievirus A16 and application thereof. The kit provided by the invention contains a fluorescein-labeled antibody, a fluorescent dye Evanslan, a slide sealing agent, a slide, a quality control slide, a cell fixing solution, a PBS solution, a PBST solution, 1% Triton-X100, a cell reference substance, a preservative sodium azide and an antibody stabilizer BSA. Through application of the kit provided by the invention, the enterovirus 71 and the coxsackievirus A16 in a sample cell can be directly detected. The kit provided by the invention takes full advantages of the direct immunofluorescence method in aspects of automation and high throughput, can achieve rapid, sensitive and efficient detection on the viruses, has an important significance in rapid and accurate clinical diagnosis and can meet different needs of large hospitals and primary hospitals.

Description

[0001] This invention is a division of the patent "a kit for direct detection of enterovirus 71 and coxsackie virus A16 and its application". The filing date of the original application is: 2016.01.21; the application number is: 201610041545.6 technical field [0002] The invention belongs to the technical field of pathogen detection, and in particular relates to a kit for directly detecting enterovirus type 71 and coxsackievirus type A16 and an application thereof. [0003] technical background [0004] Hand, foot and mouth disease (hand, foot and mouth disease, HFMD) is an infectious disease caused by enteroviruses. There are more than 20 types of enteroviruses that cause HFMD, among which coxsackievirus type A16 (CA16) and enterovirus 71 (EV71) are the most common. It mostly occurs in children under 5 years old, manifested as mouth pain, anorexia, low fever, small herpes or small ulcers in hands, feet, mouth and other parts. Complications such as meningoencephalitis. Ind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983G01N33/56966G01N2333/085
Inventor 李晨阳邓国亮李园枚邹铮铮杨伟民吴慧洁
Owner GUANGDONG HECIN SCI INC
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