Detection kit for African swine fever viruses and detection method thereof

An African swine fever virus and detection kit technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. High specificity, easy operation and low cost effect

Active Publication Date: 2020-04-03
武汉博杰生物医学科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nucleic acid hybridization technology has the characteristics of high sensitivity, strong specificity, and good practicability. It is a routine method for the diagnosis of various animal diseases, but the operation is complicated and the cost is high. Ordinary laboratories do not have corresponding equipment, and false positives are prone to occur
Gene chips and high-throughput sequencing technologies are fast and simple, with strong specificity, high sensitivity and high-throughput quantification, but they are costly and require high levels of operators

Method used

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  • Detection kit for African swine fever viruses and detection method thereof
  • Detection kit for African swine fever viruses and detection method thereof
  • Detection kit for African swine fever viruses and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 is used to detect the design and acquisition of the crRNA of African swine fever virus

[0033] 1. First design 3 specific crDNA sequences with T7 promoter according to the p72 (B646L) gene specific sequence of ASFV SY18 strain as shown in SEQ ID NO.1, and its sequences are respectively shown in SEQ ID NO.2-4 shown.

[0034] 2. Acquisition of crRNA: The reverse transcription system is shown in Table 1, and the reverse transcription condition is to incubate overnight at 37°C.

[0035] Table 1

[0036]

[0037]The crRNA was transcribed in vitro by T7 polymerase, and purified to obtain the crRNA. The sequence of the crRNA is shown in SEQ ID NO.5-7.

Embodiment 2

[0038] The detection kit and detection method of embodiment 2 African swine fever virus

[0039] 1, the composition of kit (the present invention is example with colloidal gold kit)

[0040] (1) Detection reagent:

[0041] The crRNA of African swine fever virus (as shown in any one of SEQ ID NO.5-7);

[0042] A specific fluorescent probe (sequence as shown in SEQ ID NO.8, the 3' end of the sequence is labeled with biotin, and the 5' end is labeled with cy5);

[0043] cpf1 protein;

[0044] Enzyme-free water;

[0045] DNase inhibitors.

[0046] (2) When the detection kit is a colloidal gold detection kit, such as figure 1 As shown, the colloidal gold detection kit includes a base plate 1, a sample pad 2, a bonding pad 3, a nitrocellulose membrane 4 and a water-absorbing pad 7 that are bonded to the base plate 1 and overlapped successively; A quality control line 5 is provided near the side of the binding pad 3, and a detection line 6 is provided on the side of the nitroce...

Embodiment 3

[0071] Embodiment 3 specific detection

[0072] Carry out specificity experiment according to established reaction system, use the primer of the present invention design to the plasmid DNA template of different viruses (ASFV positive plasmid, porcine blue ear virus (PRRS), porcine parvovirus (CSFV), porcine epidemic diarrhea virus (PEDV) ) positive DNA or cDNA, porcine circular virus type 2 (PCV2)) for experiments, the test results are as follows figure 2 as shown, figure 2 The results showed that except for the ASFV positive plasmid, the plasmids of several other viruses were not amplified, thus proving that the specificity of the test method was good. The electrophoresis result was consistent with the test strip result.

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Abstract

The invention provides crRNA of African swine fever virus, a detection kit and a detection method thereof. The sequence of the crRNA is shown as any one of SEQ ID NO.5 to SEQ ID NO.7. The kit comprises the crRNA, and further comprises a DNA enzyme inhibitor, cpf1 protein and an amplification system of a to-be-detected sample. A detection reagent (a CRISPR/Cpf1 detection system) composed of crRNA for detecting the African swine fever virus and RPA isothermal amplification are combined into one step, a detection result is shown in the form of a colloidal gold test strip, the operation is easy and convenient, time is short, the instruments are not required, and an isothermal reaction can be conducted. The method is simple, high in detection speed, high in sensitivity and high in specificity.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a detection kit and detection method for African swine fever virus. Background technique [0002] African swine fever (ASF) is a severe and highly contagious disease of pigs caused by African swine fever virus (ASFV). The World Organization for Animal Health (OIE) listed it as a legally notifiable animal disease, and this disease is also a class of animal epidemics that my country focuses on preventing. It is characterized by a short course of disease, the most acute and acute infection mortality rate is as high as 100%, clinical manifestations are fever (up to 40-42 ℃), rapid heartbeat, dyspnea, partial cough, serous or mucous purulence in eyes and nose Secretion, skin cyanosis, obvious bleeding in lymph nodes, kidneys, and gastrointestinal mucosa. The clinical symptoms of African swine fever are similar to those of traditional swine fever, and the syndrome is comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107C12Q2565/625
Inventor 姚杰程诚王恩慧杨欣毕延震
Owner 武汉博杰生物医学科技有限公司
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