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Combined detection card for simultaneously detecting specific IgM and IgG antibodies of ASFV as well as preparation method and application thereof

A combined detection and specific technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of high antibody detection rate and low pathogen detection rate, and achieve the effects of intuitive results, improved sensitivity, and rapid combination.

Pending Publication Date: 2021-09-03
XINGJIA BIO ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is worth noting that the study found that: the results of the challenge test of the gene deletion strain showed that the virus nucleic acid could be detected on the 28th and 49th day in cohabiting pigs, and the antibody could be detected on the 21st and 28th day; In the field detection experiment, the saliva of 20 pens of pigs was collected with cotton thread, antibodies were detected in 17 pens, but virus was detected in only 2 pens; virus detection was delayed, the detection rate of pathogens was low and the detection rate of antibodies was high

Method used

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  • Combined detection card for simultaneously detecting specific IgM and IgG antibodies of ASFV as well as preparation method and application thereof
  • Combined detection card for simultaneously detecting specific IgM and IgG antibodies of ASFV as well as preparation method and application thereof
  • Combined detection card for simultaneously detecting specific IgM and IgG antibodies of ASFV as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0057] Such as Figure 1~3 As shown, a combined detection card for simultaneously detecting ASFV-specific IgM and IgG antibodies in this embodiment includes a cartridge 1, and an IgM test strip 2 and an IgG test strip 3 are arranged in parallel inside the cartridge 1. The IgM detection strip 2 and the IgG detection strip 3 respectively include a bottom plate 4, and on each bottom plate 4, according to the flow and migration direction of the sample to be tested, a sample pad 5, a sample binding pad 6, a nitrocellulose membrane 7 and an absorbent pad are arranged in sequence. 8; wherein, the absorbent pad 8 partially overlaps the nitrocellulose membrane 7, the nitrocellulose membrane 7 partially overlaps the sample binding pad 6, and the sample binding pad 6 partially overlaps the sample pad 5.

[0058] Specifically, in this embodiment, the nitrocellulose membrane 7 of the IgM test strip 2 is respectively provided with C M Quality control line 21 and T 1M Detection line 22, T ...

Embodiment 2

[0062] A method for preparing a joint detection card for simultaneously detecting ASFV-specific IgM and IgG antibodies in a kind of above-mentioned embodiment 1 of the present embodiment comprises the following steps:

[0063] (1) Preparation of biotinylated antibody

[0064] Take 100 μg goat anti-pig IgM and mouse anti-pig IgG antibodies respectively, add biotin according to the ratio of antibody / biotin molar ratio of 1:100, and the total volume is 100 μL; incubate at room temperature for 4 hours, and dilute to the reaction solution with PB buffer 5 times the volume, and dialyzed at 4°C for 72 hours to obtain biotin-labeled anti-pig IgM and biotin-labeled anti-pig IgG.

[0065] (2) Preparation of colloidal gold-labeled streptavidin

[0066] Add 2mL of 0.5% trisodium citrate solution to 50mL of boiling 0.01% chloroauric acid aqueous solution, and then add 0.1mol / L K 2 CO 3 Adjust the colloidal gold pH to 8.5 to obtain a stable gold sol;

[0067] Take 100mL of colloidal go...

Embodiment 3

[0086] Functional verification of the combined detection card for simultaneous detection of ASFV-specific IgM and IgG antibodies in a kind of above-mentioned embodiment 1 of the present embodiment:

[0087] Take 9 reagent cards prepared above and place them flat on a dry surface, add 10 μL of ASFV wild virus positive serum (purchased from China Veterinary Microorganism Culture Collection Management Center) directly above the sample hole on the test paper card, and drop 10 μL of ASFV wild virus positive serum on 3 Add 10 μL ASFV gene deletion virus positive serum (purchased from China Veterinary Microbiology Culture Collection Management Center) directly above the sample hole of the test paper card, and add 10 μL normal pig serum (purchased) directly above the sample hole of the test paper card to the other 3 tubes As a negative control, add 2 drops of buffer solution vertically to the sample well, time it for 5 minutes, and judge the test result according to the result judgment...

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Abstract

The invention provides a combined detection card for simultaneously detecting specific IgM (Immunoglobulin M) and IgG (Immunoglobulin G) antibodies of ASFV (African Swine Fever Virus). An IgM detection strip and an IgG detection strip are combined on one detection card. Each detection strip comprises a sample pad, a sample combination pad, a chromatography membrane and a water absorption pad. The two chromatography membranes adopt the same recombinant ASFV antigens as solid phase coating substances, namely MGF 360-14L, CD2v and p72. The sample combination pad of the IgM detection strip is coated with biotin-labeled anti-pig IgM, a tracer-labeled streptavidin and a tracer-labeled rabbit anti-chicken IgY; and the sample combination pad of the IgG detection strip is coated with biotin-labeled anti-pig IgG, a tracer-labeled streptavidin and a tracer-labeled rabbit anti-chicken IgY. The invention also provides a preparation method and application of the combined detection card. The detection card is prepared by using an immunochromatography technology, the operation is convenient, and the result is visual.

Description

technical field [0001] The invention relates to the field of in vitro diagnosis and biomedical detection reagents, in particular to a combined detection card for simultaneously detecting ASFV-specific IgM and IgG antibodies, a preparation method and application thereof. Background technique [0002] On March 22, 2021, the website of the Ministry of Agriculture and Rural Affairs released the "Technical Guidelines for Monitoring African Swine Fever (ASFV) Variant Strains in Pig Farms". This guideline is the National African Swine Fever Reference Laboratory. In order to guide pig farms to do a good job in the monitoring of African swine fever, to detect African swine fever virus in time, especially for the current "vaccine virus" infection caused by illegal vaccines, to improve the prevention and control of African swine fever. It is specially designed for control ability. [0003] African swine fever mutant strains include gene deletion strains, natural mutant strains, natura...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/558
CPCG01N33/56983G01N33/558
Inventor 黄逸强戈军陈娟彭湘平李尤美
Owner XINGJIA BIO ENG CO LTD
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