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African swine fever virus antibody colloidal gold duplex detection test strip and preparation method thereof

A technology for detection of African swine fever virus and test strips, which is applied in the field of African swine fever virus antibody colloidal gold dual detection test strips and its preparation, and can solve the problem that the color difference between the detection line and the quality control line of positive samples and negative samples is not obvious , It is impossible to realize the whole process monitoring of African monkey swine fever antibody, and the color difference between the detection line and the quality control line is not obvious, so as to achieve the effect of simplifying steps and time, good specificity and high accuracy

Inactive Publication Date: 2021-07-09
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing colloidal gold test strips for p30 protein and / p72 protein have the following problems: ① the existing African swine fever colloidal gold test strips can only detect p30 protein and / p72 protein alone, and cannot achieve African monkey swine fever Whole-process monitoring of antibodies; ② Even if conventional methods are used to prepare test strips for detecting p30 protein and / or p72 protein, due to the use of conventional gold-labeled protein protectant and gold-labeled protein blocking solution in the preparation process of the binding pad, positive samples and The color difference between the detection line and the quality control line of negative samples is not obvious, which affects the judgment of the result; ③The use of conventional sample pad blocking solution and sample diluent will also cause the color difference between the detection line and quality control line of positive samples and negative samples Not obvious, affecting the judgment of the result

Method used

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  • African swine fever virus antibody colloidal gold duplex detection test strip and preparation method thereof
  • African swine fever virus antibody colloidal gold duplex detection test strip and preparation method thereof
  • African swine fever virus antibody colloidal gold duplex detection test strip and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] The ASFVΔ181 / UK attenuated strain described in the following examples was obtained from the African Swine Fever Regional Reference Laboratory (Lanzhou). Preparation of embodiment 1p30 protein and p72 protein

[0035] 1. Preparation of p30 recombinant protein

[0036] Transform the recombinant positive plasmid pET-32a-p30 into competent cells BL21(DE3), culture overnight at 37°C; pick a single colony and inoculate it in LB medium containing ampicillin (100 μg / ml), and culture at 37°C for 12 hours , as seeds; the cultured seeds were inoculated in LB medium containing ampicillin (100 μg / ml) at a ratio of 1:100, and cultured at 37°C until OD 600 When the nm is about 0.65, add IPTG to a final concentration of 0.5mmol / L, and continue to culture at 37°C for 6-8h; collect the bacteria by centrifugation, resuspend the bacteria with an appropriate amount of buffer, sonicate, centrifuge at 12000r / min for 10min, discard Remove the supernatant, wash 4-6 times with PBS, dissolve th...

Embodiment 2

[0039] Embodiment 2 Preparation of African swine fever virus antibody colloidal gold double detection test strip

[0040] 1. Preparation of Conjugated Pads

[0041] 1.1 Preparation of colloidal gold solution

[0042] Add 1ml 1% chloroauric acid in 100ml ultrapure water and mix well, heat until the bubbles appear evenly and rise upwards, then add 2.0ml 1% trisodium citrate solution at one time, heat until the color After no change, continue heating for 2-3 minutes, carefully remove the Erlenmeyer flask, let it cool to room temperature, and add ultrapure water to make up to 100ml.

[0043] 1.2 Preparation of gold-labeled protein

[0044] (1) Optimum marked pH value

[0045] Use 0.1mg / ml K 2 CO 3 Solution Adjust the pH of the gold solution to 4-9 respectively, add appropriate amount of protein to the gold solution with different pH, let it stand at room temperature for 2 hours, centrifuge at 12000r / min for 30 minutes at 4°C, collect the supernatant and coat the ELISA plate, ...

Embodiment 3

[0092] Example 3 Use and result judgment of African swine fever virus antibody colloidal gold double detection test strip

[0093] Dilute the sample to be tested 1:10 with the sample diluent, drop 100 μl of the sample on the sample pad at room temperature and observe the result within 5-10 minutes; the judgment result is as follows: Figure 5 As shown, if both the test line (T line) and the quality control line (C line) appear red, it is positive; if the test line (T line) does not show color, but the quality control line (C line) shows color, then it is negative ; If the quality control line (C line) does not develop color, it can be judged that the test strip is invalid.

[0094] Detection and determination principle: Add the sample to be tested to the sample pad, and it will be chromatographically forward through capillary action. When it reaches the binding pad, if the sample to be tested contains p30 and / or p72 antibodies, the protein on the binding pad will correspond to...

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Abstract

The invention discloses a colloidal gold duplex detection test strip for whole-course monitoring of African swine fever virus antibodies and a preparation method thereof. The test strip comprises a PVC base plate, wherein the PVC base plate is sequentially provided with a sample pad, a combination pad, a nitrocellulose film and a water absorption pad from left to right, the nitrocellulose film is provided with a detection line and a quality control line, the combination pad is coated with ASFV p30 protein and ASFV p72 protein which are labeled by colloidal gold, the detection line is coated with ASFV p72 and ASFV p30 protein, and the quality control line C is coated with an anti-ASFV p30 protein monoclonal antibody and / or an anti-ASFV p72 protein monoclonal antibody; by optimizing the preparation of the combination pad, the whole-process monitoring of the African swine fever virus antibody is realized, compared with a single p30 or p72 test strip, the infection period of an organism can be more accurately obtained, and meanwhile, the combination is good in specificity and relatively high in sensitivity and is expected to become a technical means for on-site or laboratory detection of the African swine fever virus antibody; and a new idea is provided for accurate and rapid diagnosis of African swine fever.

Description

technical field [0001] The invention relates to the technical field of rapid immunochromatographic detection, in particular to an African swine fever virus antibody colloidal gold double detection test strip and a preparation method thereof. Background technique [0002] African swine fever (ASF) is an acute, virulent, highly contagious infectious disease caused by African swine fever virus (ASFV). ASF was first discovered in Kenya in 1920. Since the first case of African swine fever in my country in August 2018, it has spread rapidly across the country. ASFV particles have an icosahedral symmetrical structure with a diameter of about 200 nm. It is a large enveloped double-stranded DNA virus. The virus is not only the only member of the Asfivirus family Asfarviridae, but also the only known DNA arbovirus. The genome length ranges from about 170 to 193kb, encoding more than 160 open reading frames (ORFs). Studies have shown that more than 50 proteins are packaged into virio...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543
CPCG01N33/54313G01N33/56983
Inventor 郑海学田宏万颖石正旺罗俊聪彭高闯王丽娟茹毅麻园周改静宋锐
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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