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African swine fever virus nucleic acid rapid detection kit and application

A technology for African swine fever virus and swine fever nucleic acid, which is applied in the field of kits for rapid detection of African swine fever virus nucleic acid, can solve the problems affecting the amplification speed and detection sensitivity, the difficulty of primer design, and the impact on on-site detection. The effect of short time, good specificity and accurate detection means

Inactive Publication Date: 2021-05-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Because the RPA method is very different from the traditional PCR method, it has higher requirements for primers and probes, which is the key to affecting the RPA method. Different primers directly affect the amplification speed and detection sensitivity, thereby affecting on-site detection, and There is currently no dedicated RPA primer design software
Therefore, the primer design of RPA is more difficult

Method used

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  • African swine fever virus nucleic acid rapid detection kit and application
  • African swine fever virus nucleic acid rapid detection kit and application
  • African swine fever virus nucleic acid rapid detection kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 specific detection

[0032] 1. Primer Design

[0033]According to the design principle of RPA primers, the primer length is 30-35bp, design several pairs of specific primers, label the 5' end of the upstream primer with fluorescein isothiocyanate, and label the 5' end of the downstream primer with biotin, and the amplified fragment size is 292bp , using the recombinant plasmid PμCsp-ASFV-P72 DNA as a template for RPA amplification, through the test strip detection of the amplified product, and evaluating and screening the amplification efficiency and specificity of the primers according to the specificity of the amplified product, so as to select Best Primer.

[0034] The present invention has designed and synthesized several primers, and the primer sequences screened in this example are shown in Table 1, wherein multiple primers are designed, and the primer sequences finally selected after screening are shown in the sequence table SEQ ID NO 1~SEQ ID NO 2 ...

Embodiment 2

[0050] Embodiment 2 sensitivity test

[0051] copy number is 10 6 、10 5 、10 4 、10 3 、10 2 、10 1 The PμCsp-ASFV-P72 plasmid DNA was used as a template, and RPA amplification was performed according to the conditions of Example 1. The amplified products were detected by test strips, and the results were as follows: image 3 shown. PμCsp-ASFV-P72 recombinant plasmid DNA copy number is 10 6 、10 5 、10 4 、10 3 、10 2 、10 1 copies / μL (numbered 1, 2, 3, 4, 5, 6), and number 7 is a blank control. The results show that bands can still be detected when the copy number is as low as 100 copies / μL.

[0052] Embodiment 3 test strip

[0053] The invention provides a colloidal gold test strip for detecting RPA nucleic acid amplification products, which includes a gold label pad, a detection line and a quality control line, and the gold label pad is adsorbed with an anti-FITC gold label antibody. The coating concentration of the gold-labeled antibody is (1 mg / L) and the coating vo...

Embodiment 4

[0054] Application of embodiment 4 African swine fever virus nucleic acid rapid detection test strip

[0055] In this embodiment, the positive sample is a DNA sample of pig tissue infected with ASFV, and the negative sample is a DNA sample of normal pig tissue. Using ASFV-positive DNA samples and ASFV-negative DNA samples as templates, RPA amplification was carried out. The RPA amplification reaction procedure was: constant temperature reaction at 39°C, amplification for 20 minutes, and the amplification products were detected by test strips. The results were as follows: Figure 4 shown. Numbers 1-3 are three ASFV-positive DNA samples, 4-6 are three ASFV-negative DNA samples, and number 7 is a blank control. The results show that bands appear on the T and C lines of the test strip in samples 1-3, and only appear on the C line in samples 4-7, indicating that the sequences of SEQ ID NO 1~SEQ ID NO 2 are available testing on real samples.

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Abstract

The invention provides an African swine fever virus nucleic acid rapid detection method and kit, and belongs to the technical field of biology. The kit provides specific RPA labeled primers ASF-F and ASF-R for detecting nucleic acid of the African swine fever virus, and the sequences of the specific RPA labeled primers ASF-F and ASF-R are shown as SEQ ID No.1 and SEQ ID No.2. The kit also provides a detection test strip which can be used for detecting RPA amplification products. The RPA primers are adopted to amplify sample DNA respectively, the amplification products are detected through the test strip, and whether the sample is infected with the African swine fever virus or not can be judged. The detection method established aiming at the nucleic acid of the African swine fever virus does not need special equipment, the reaction time is only 25 minutes, the sensitivity is up to 100 cp / microliter, no cross reaction exists in four common swine-origin viruses, and the specificity is high. The African swine fever virus nucleic acid rapid detection kit provides a simple and effective detection means for detecting the nucleic acid of the African swine fever virus, and is suitable for field detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for rapidly detecting African swine fever virus nucleic acid. Background technique [0002] African swine fever (ASF) is a severe and highly contagious disease of pigs caused by African swine fever virus (ASFV). ASF has the characteristics of high morbidity and mortality, and once it occurs, the economic loss will be huge. In view of the serious harm of the disease, the World Organization for Animal Health (OIE) listed it as a legally notifiable animal disease, and my country listed it as a first-class animal disease. On August 1, 2018, my country confirmed the first case of African swine fever. [0003] At present, there is no effective commercial vaccine for the prevention and control of ASF. The control of ASF can only rely on laboratory diagnosis, killing of diseased animals, effective quarantine measures and strict sanitation measures. Therefore, the rapid detection of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101
Inventor 刘榜付明周翔张庆德
Owner HUAZHONG AGRI UNIV
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