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Fluorescence detection test strip for canine coronavirus antibody as well as preparation method and application thereof

A coronavirus and fluorescence detection technology, applied in the field of canine coronavirus antibody detection, can solve the problems of high test conditions and operation level, limit the prevalence of canine coronavirus, narrow detection range, etc., to improve the binding efficiency, high practical value and The effect of promotion value and short detection time

Active Publication Date: 2019-05-17
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The hemagglutination inhibition test requires fresh and highly sensitive red blood cells, and the detection time takes 2 to 3 hours; the neutralization test is cumbersome to operate, requires high test conditions and operating levels, and the whole test takes 1 to 2 weeks to produce results; ELISA The method has high requirements for operation and needs the results of the microplate reader, and is not suitable for the detection of a single sample; although the immunocolloidal gold method is convenient for detection and does not require professional technicians and working environment, its detection sensitivity is low, and it can only be qualitative. Narrow detection range
These all limit their application in the clinical detection of canine coronavirus antibody. Therefore, the purpose of the patent of the present invention is to overcome the shortcomings of the above methods and to establish a quantitative detection of canine coronavirus antibody that is easy to operate, sensitive, specific and fast. Method, applied to the monitoring of canine coronavirus immune effect, thereby limiting the prevalence of canine coronavirus

Method used

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  • Fluorescence detection test strip for canine coronavirus antibody as well as preparation method and application thereof
  • Fluorescence detection test strip for canine coronavirus antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the composition of canine coronavirus antibody fluorescence detection test strip ( figure 1 )

[0028] The test strip is composed of a bottom plate (8), a sample absorbent pad (1), a conjugate release pad a (2), a conjugate release pad b (3), a nitrocellulose membrane (4), an absorbent pad (5) composition;

[0029] The sample absorbent pad (1), conjugate release pad a (2), conjugate release pad b (3), nitrocellulose membrane (4), and water absorbent pad (5) are pasted on the PS bottom plate (8) in sequence Above; 1 / 4 area of ​​the conjugate release pad a (2) from the starting end is covered by the sample absorbent pad (1), and 1 / 4 area of ​​the conjugate release pad b (3) from the starting end is covered by the conjugate release pad a ( 2) Covering, the end of the conjugate release pad b (3) is connected to the beginning of the nitrocellulose membrane (4), the end of the nitrocellulose membrane (4) is connected to the beginning of the absorbent pad (5), ...

Embodiment 2

[0031] Embodiment 2: the preparation of test strip described in embodiment 1

[0032] The preparation method of this test strip mainly comprises the following steps:

[0033] 1) Prepare a nitrocellulose membrane with a detection line coated with canine coronavirus N expression protein and a quality control line coated with sheep anti-chicken anti-antibody;

[0034] 2) Prepare conjugate release pad a sprayed with chicken anti-dog anti-antibody-biotin conjugate and conjugate release pad b sprayed with streptavidin-fluorescent microsphere marker;

[0035] 3) Fix the sample absorbent pad, conjugate release pad a, conjugate release pad b, nitrocellulose membrane, and water absorbent pad on the base plate in sequence.

[0036] The following step-by-step detailed description:

[0037] (1) Preparation of each component

[0038] 1. Expression and purification of canine coronavirus N protein

[0039] (1) Partial gene sequence selection of CCV N protein

[0040] ①DNA序列:ATGAATACTATACCT...

Embodiment 3

[0076] Embodiment 3: the use of test strip described in embodiment 1

[0077]1. Sample Preparation

[0078] (1) Plasma samples: collect blood with an anticoagulant tube, or add EDTA anticoagulant to the blood collection tube first, add the collected blood and shake it slowly, and centrifuge at 3000-5000r / min for 10-30min at room temperature (adjust according to the amount of blood collected). The upper layer of yellow clear liquid is drawn as the plasma sample.

[0079] (2) Serum sample: Add the collected blood into a blood collection tube (without anticoagulant), place it at an incline at room temperature, and centrifuge at 3000-5000 r / min for 10-30 minutes at room temperature when a yellow clear liquid is precipitated on the upper layer (adjust according to the amount of blood collected). The upper layer of clear yellow liquid is drawn as the serum sample.

[0080] (3) Sample dilution: Take the above-mentioned sample that has been returned to room temperature, accurately p...

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Abstract

The invention discloses a fluorescence detection test strip for a canine coronavirus antibody as well as a preparation method and application thereof. The test strip provided by the invention comprises a sample absorption pad, a conjugate release pad a, a conjugate release pad b, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein a chicken anti-dog anti-antibody-biotin conjugate is sprayed on the conjugate release pad a; a streptavidin-fluorescent microsphere marker is sprayed on the conjugate release pad b; a detection line and a quality control line are arranged onthe nitrocellulose membrane; the detection line is coated with a canine coronavirus N expression protein; and the quality control line is coated with a sheep anti-chicken anti-antibody. The test strip disclosed by the invention has the advantages of high sensitivity, high specificity, easiness in operation, economical efficiency and practicability, and can realize field rapid quantitative detection of the canine coronavirus antibody.

Description

technical field [0001] The invention relates to the detection of canine coronavirus antibody, in particular to a canine coronavirus antibody fluorescence detection test strip and its preparation method and application. technical background [0002] Canine coronavirus infection is a highly contagious disease caused by canine coronavirus (CCV), with acute gastroenteritis in dogs as the main symptom, characterized by frequent vomiting, diarrhea, depression and anorexia. At present, two genotypes of CCV have been identified, namely CCV-I and CCV-II. In general, CCV causes diarrhea of ​​variable severity in dogs, and co-infection with other pathogens in dogs can lead to fatal outcomes. The disease is distributed worldwide, and there are also reports of military and police dogs and civilian dogs in my country. With the successful development and wide application of canine vicious infectious disease vaccines such as canine distemper and canine parvovirus enteritis in China, the h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569G01N33/531
Inventor 何方洋吴小胜申梁万宇平朱亮亮鲁晓雄李斌冯才伟崔海峰刘玉梅
Owner BEIJING KWINBON BIOTECH
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