Freeze-dried microchip, kit and method for detecting African swine fever virus

An African swine fever virus and microchip technology, which is applied in the field of freeze-dried microchips for detecting African swine fever virus in pig tissue and pig blood feed products, can solve the problems of cumbersome operation, long detection time, and easy pollution, and achieve Good repeatability, consumption saving and high degree of automation

Pending Publication Date: 2020-07-28
CHINA ANIMAL DISEASE CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used ASF virus nucleic acid detection methods are PCR and Realtime PCR; while the traditional ASF isolation and identification methods are banned in my country due to potential biological safety hazards
Each of the above methods has disadvantages such as cumbersome operation, long detection time and easy pollution.

Method used

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  • Freeze-dried microchip, kit and method for detecting African swine fever virus
  • Freeze-dried microchip, kit and method for detecting African swine fever virus
  • Freeze-dried microchip, kit and method for detecting African swine fever virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Preparation of freeze-dried microchip

[0046] This group of embodiments provides a freeze-dried microchip for detecting African swine fever virus in pig tissues and pig blood feed products, which is characterized in that a fluorescent PCR reaction system for detecting African swine fever virus is fixed in place by freeze drying. On the microchip;

[0047] The fluorescent PCR reaction solution includes the following primers and probes:

[0048] F (Sequence 1 of the Sequence Listing): 5'-CAGATGCCGATACCACAAG-3';

[0049] R (sequence 2 of the sequence listing): 5'-GAGGAATACCAACCCAGTG-3';

[0050] P (sequence 3 of the sequence listing): 5'-CAGCCGTAGTGATAGACCCCAC-3'.

[0051] In a specific embodiment, the fluorescent PCR reaction system further includes: Taq enzyme, trehalose, Tris-Cl, dNTP, Mg 2+ ;

[0052] Preferably, the 5'end of the Taqman probe of the African swine fever virus is labeled with a fluorescent reporter group; the 3'end is labeled with a fluorescence quenchin...

Embodiment 2

[0066] Example 2: Preparation of the kit

[0067] This group of embodiments provides a freeze-dried microchip fluorescent PCR kit for detecting African swine fever virus in pig tissues and pig blood feed products, including the freeze-dried microchip described in any one of the first group of embodiments.

[0068] In a further embodiment, the kit further includes: a diluent for dropping into the sample hole of the freeze-dried microchip, and then placing the freeze-dried microchip on a fluorescent PCR machine for fluorescent PCR amplification.

[0069] The diluent is specifically PCR 10×buffer, and the specific components do not contain Mg 2+ , Containing 500mM KCl, 100mM Tris-HCl, 0.1% gelatin, the dilution is commercially available. When using, you need to use nuclease-free water to dilute the diluent into 2×buffer, and add 0.6μL to each well of the microchip.

[0070] In a further embodiment, the kit further includes: mineral oil, which is used to seal the sample hole on the microc...

Embodiment 3

[0078] Example 3: Lyophilized microchip fluorescent PCR method for detecting African swine fever virus in pig tissues and pig blood feed products

[0079] This group of embodiments provides a freeze-dried microchip fluorescent PCR detection method for detecting African swine fever virus in feed. All the embodiments in this group have the following characteristics: the method includes: using the freeze-dried microchip described in any one of embodiment 1, and / or, performing fluorescent PCR amplification of the test sample with the kit described in any one of embodiment 2 increase.

[0080] In some embodiments, after adding the sample to be tested and the diluent into the sample hole of the freeze-dried microchip, the freeze-dried microchip is placed on a fluorescent PCR machine to perform the fluorescent PCR amplification;

[0081] Preferably, the added amount of the diluent 2×buffer is 0.6 μL, and the added amount of the positive control, negative control or sample is 0.6 μL;

[0082...

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Abstract

The invention discloses a freeze-dried microchip, a kit and a method for detecting African swine fever virus in pig tissue and pig blood feed products. The freeze-dried microchip is characterized in that a fluorescent PCR reaction system for detecting the African swine fever virus is fixed on the microchip through freeze-drying; the kit comprises a freeze-dried microchip, a bottle of mineral oil,a tube of positive control, a tube of negative control, a tube of diluent and a tube of nuclease-free water. A primer probe group is used for identifying the African swine fever (ASF) virus, and detecting whether to-be-detected pig tissue and a pig blood feed product sample contain the African swine fever virus or not. By utilizing the freeze-dried microchip, the kit and the method provided by theinvention, ASF virus is detected from pig tissue and pig-derived feed for the first time, it is proved that the freeze-dried microchip has an important application value on prevention and control ofthe ASF, is beneficial to finding and cutting off a virus transmission chain, and can effectively prevent large-scale outbreak.

Description

Technical field [0001] The invention relates to the field of animal virus detection, in particular to a freeze-dried microchip, kit and method for detecting African swine fever virus in pig tissues and pig blood feed products. Background technique [0002] African Swine Fever (ASF) is an infectious disease caused by a virus, which can affect pigs of all ages and cause hemorrhagic fever. The fatality rate in acute onset can reach 100%. African swine fever is a serious threat to the production of the pig industry, and the epidemic has triggered trade restrictions, which has a major impact on international trade. ASF has strong cross-border communication capabilities and has attracted more and more attention from governments and international organizations. my country has never found ASF before August 2018, and has always listed it as a key foreign animal disease to prevent. [0003] From August to late September 2018, my country has successively operated 20 sites in 8 provinces inc...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6837C12N15/11
CPCC12Q1/701C12Q1/6837
Inventor 倪建强王传彬刘洋杨林徐琦宋晓晖顾小雪李硕刘玉良亢文华
Owner CHINA ANIMAL DISEASE CONTROL CENT
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