Fluorescent quantitative PCR on-site rapid detection kit for African swine fever virus

A technology for African swine fever virus and detection kits, which is applied in the field of on-site rapid detection kits for African swine fever virus fluorescence quantitative PCR, can solve the problems of many steps in the extraction process, large differences in test results, and many types of reagents, etc., and achieve reduction Time and operation steps, suitable for transportation and storage, and the effect of saving detection time

Pending Publication Date: 2019-12-31
青岛立见生物科技有限公司
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  • Abstract
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Problems solved by technology

[0004] At present, fluorescent quantitative PCR detection kits are relatively commonly used on-site rapid detection methods. Viral nucleic acid extraction methods include column extraction method or magnetic bead method. These methods extract viral nucleic acid with high purity, but at the same time there are many shortcomings: 1) The extraction process of viral nucleic acid has many steps and takes a long time; 2) There are many types of instruments used for extraction, which are not suitable for on-site rapid detection of large enterprises or breeding industries; 3) There are many types of reagents used for extraction, which are inconvenient to use. It is easy to make mistakes; 4) Reagents such as ethanol are used in the extraction, and there are potential safety hazards in transportation and use; 5) The extraction has high requirements for operators, and a lot of training needs to be carried out for grassroots personnel; 6) The sensitivity of the extraction method is too high, and it is easy to lead to large discrepancies in test results

Method used

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  • Fluorescent quantitative PCR on-site rapid detection kit for African swine fever virus
  • Fluorescent quantitative PCR on-site rapid detection kit for African swine fever virus
  • Fluorescent quantitative PCR on-site rapid detection kit for African swine fever virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Detection of clinical whole blood samples

[0027] A fluorescent quantitative PCR detection kit for African swine fever virus, said kit comprising: buffer B1, buffer B2, PCR reaction solution, positive control and negative control;

[0028] The buffer B1: guanidine isothiocyanate 1-6M, sodium chloride 0.1-3M, ethylenediaminetetraacetic acid 0.01-0.5M, adjust the pH value to 8.5-12.7;

[0029] The components of the buffer B2 are: ammonium acetate 0.1-1.0M, Tris-Cl 0.01-1M, and the pH value is adjusted to 7.5-8.5.

[0030] The PCR reaction solution: composed of 2×Premix Ex Taq TM Composed of enzyme mixture, ASFV-specific upstream and downstream primers and fluorescent probes, Tli RNaseH is added to the enzyme mixture; Tli RNaseH is a heat-resistant RNaseH, which can be well eliminated when DNA is used as a template for PCR reactions The primers and probe sequences of the obstacles that inhibit the PCR reaction are as follows:

[0031] Upstream primer: 5'-...

Embodiment 2

[0052] Embodiment 2: Detection of clinical tissue samples

[0053] The kit used in this example is the same as Example 1.

[0054] 1) Pretreatment of tissue samples

[0055] Collect 3 samples of spleen, 3 lymph nodes, and 4 muscle tissue samples, weigh 0.1-0.2g- (soy bean size) for each sample, add 10 times the volume of about 1-2ml normal saline (0.9% sodium chloride), Make a tissue homogenate, centrifuge at 10,000 rpm for 1 minute, and take the supernatant for the next step of nucleic acid extraction.

[0056] 2) Acquisition of sample DNA virus nucleic acid:

[0057] (1) Buffer B1 Lysis: First take a 1.5ml centrifuge tube and add 100 μL of buffer B1, take 10 samples of tissue homogenate that have been processed in the above steps, add 20 μL of each sample to buffer B1 and mix well, vortex at room temperature Rotate and shake to mix 3-5 times, lyse at room temperature for 3 minutes.

[0058] (2) Buffer B2 to terminate the lysis: add 100 μL of buffer B2 to the above mixtur...

Embodiment 3

[0069] Embodiment 3: The impact of different DNA virus genome extraction methods on ASFV detection

[0070] There are many types of DNA extraction kits currently on the market, such as column extraction method, magnetic bead method, etc. In this embodiment, the ASFV nucleic acid extracted by the common column extraction method and the magnetic bead method and the corresponding ASFV fluorescence quantitative PCR method are compared with the ASFV fluorescence quantitative PCR rapid detection kit based on nucleic acid extraction-free direct amplification developed by the present invention. To explore the influence of different DNA virus nucleic acid extraction methods on ASFV detection. In this example, two ASFV clinically positive serum samples were selected as samples to be tested, and after DNA was extracted by three methods, fluorescent PCR reaction was carried out. The detection steps are as follows:

[0071] 1. Extraction of DNA from samples

[0072] Method 1. Extraction...

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Abstract

The present invention relates to a fluorescent quantitative PCR detection kit for African swine fever viruses (ASFV) and belongs to production and technical field of veterinary biological diagnosis products. The kit comprises the following components: a buffer B1, a buffer B2, a PCR reaction solution, a positive control and a negative control; the buffer B1 quickly cracks samples to release virusnucleic acid; the buffer B2 is used for stabilizing the extracted viral nucleic acid and eliminating an inhibition effect on PCR reaction; and PCR reaction solution is composed of a 2xPremixExTaqTM enzyme mixed solution, ASFV specific upstream and downstream primers and a fluorescent probe, wherein Tli RnaseH is added into the enzyme mixed solution. The two buffer systems easy for cracking are used to directly crack nucleic acid DNA in the samples, greatly reduce time and operation steps for extracting the nucleic acid DNA in the samples, save detection time and are also suitable for transportation and storage.

Description

technical field [0001] The invention belongs to the production and technical field of veterinary biological diagnostic products, and relates to the development and application of a rapid detection kit for African swine fever virus fluorescence quantitative PCR, in particular to a method for rapidly detecting African swine fever virus nucleic acid in pig and pork products Development and application of rapid detection kits. Background technique [0002] African Swine Fever (ASF) is an acute, febrile, highly contagious zoonotic disease caused by African Swine Fever Virus (ASFV), with a rapid onset and a fatality rate of over 90%. , Seriously endanger the development of pig industry. The World Organization for Animal Health (OIE) lists it as a legally notifiable disease, and it is listed as a first-class infectious disease in my country. On August 3, 2018, my country's first ASF outbreak occurred in a pig farm in Shenyang City. Since then, more than 100 African swine fever ou...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2563/107
Inventor 孙学强宫枫举官丽娟李翠翠张志
Owner 青岛立见生物科技有限公司
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