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African swine fever virus detection reagent kit for environment samples and detection method

A technology of African swine fever virus and detection kit, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems that cannot meet the actual needs of African swine fever recovery breeding

Active Publication Date: 2020-06-02
广州赛百纯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The Chinese patent with the application number 201910853261.0 discloses that the extraction-free method can only detect diseased pigs or endangered pigs, which cannot meet the actual needs of African swine fever recovery breeding

Method used

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  • African swine fever virus detection reagent kit for environment samples and detection method
  • African swine fever virus detection reagent kit for environment samples and detection method
  • African swine fever virus detection reagent kit for environment samples and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Take 3 soil samples from around the breeding plant of Shangyuan Ecological Agriculture Company, respectively numbered 19001, 19002, and 19003; 1 sample from the bottom of the manger in the pig house (containing feed residues, soil, and feces), sample number 19004; 2 samples containing pig saliva Pack (Guangdong Aisu Biotechnology Co., Ltd.) to take the sample back and squeeze out the saliva sample, collect it in a 1.5ml centrifuge tube, sample number: 19005, 19006; sample 3 pig feeds from Guangzhou Haid Feed Co., Ltd., sample number 19007, 19008, 19009; 3 water samples were taken from the surrounding areas of the breeding base of Muyuan Co., Ltd., and the sample numbers were 190010, 190011, and 190012. The DNA extraction reagent (A) in the environmental sample African swine fever virus detection kit according to the present invention and the purchased virus DNA & RNA extraction reagent (Zhengzhou Zhongdao Biotechnology Co., Ltd.) were respectively used for sample process...

Embodiment 2

[0074] Use environmental sample African swine fever virus nucleic acid extraction reagent of the present invention to swine fever virus, porcine parvovirus, porcine pseudorabies virus, porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine rotavirus (G5 type) , porcine circovirus type 2 (ZJ / C), Streptococcus suis, Escherichia coli, porcine blue ear virus, foot-and-mouth disease (A\O\Asian type) virus is extracted, extracts nucleic acid and adopts environmental sample African pig of the present invention Pestivirus detection kit and detection method amplification detection, the specific operation steps are the same as in Example 1, and the results are as follows figure 2 and shown in Table 4.

[0075] Experimental results:

[0076] Table 4 Experimental results of different virus detection

[0077]

[0078] Depend on figure 2 As can be seen from the experimental results in Table 4, the detection of the above-mentioned viruses is negative wi...

Embodiment 3

[0080] According to the African swine fever virus sequence reported by NCBI, clone the positive control plasmid DNA (the nucleotide sequence of the positive control is such as SEQ ID NO: 4), dilute according to the 10-fold gradient, and perform the dilution according to the relationship between concentration and Copise number extrapolated to a single copy. The obtained gradient DNA is amplified, and the sensitivity of the test reagent is tested. The specific operation steps of its detection are the same as in Example 1, and the detection results are as follows: image 3 shown. image 3 It is the positive plasmid of serial dilution, and the copy value of the dilution after quantification reflects the sensitivity of the test kit.

[0081] Experimental results: Y=-3.273*LOG(X)+40.19, R2=1, the amplification efficiency is 102.1%, the linear correlation coefficient of the gradiently diluted plasmid is R2=1, the negative control has no Ct, the experiment is valid, and the product a...

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Abstract

The invention relates to an African swine fever virus detection reagent kit for environment samples and a detection method, and belongs to the technical field of biology. The African swine fever virusdetection reagent kit for environment samples provided by the invention comprises specific primers AP72-F ( SEQ ID NO:1 ) and AP72-R ( SEQ ID NO:2 ), and a TaqMan probe AP72-P ( SEQ ID NO:3 ), wherein 5' end marked report fluorescent dye of the probe is FAM, and a 3' end marked fluorescent quenching group is BHQ-1. The African swine fever virus detection reagent kit for environment samples and the detection method provided by the invention can be widely used for detecting various environment samples in breeding environment, the limitation for detecting a single environment sample is broken, the sensitivity of the detection method reaches 10copise / [mu]l, the detection of African swine fever viruses of the environment samples does not have a cross reaction with various viruses, and the African swine fever virus detection reagent kit for environment samples and the detection method can be widely suitable for real-time monitoring of rehabilitation and breeding of pigs suffering from African swine fever.

Description

technical field [0001] The invention relates to an environmental sample African swine fever virus detection kit and a detection method, belonging to the field of biotechnology. Background technique [0002] The prevention and control of African swine fever virus has always been a key epidemic prevention and control in various countries. Whether it is Spain, Brazil and other countries that have successfully eradicated African swine fever, or Russia, which is still struggling with African swine fever, historical experience has proved that it takes a lot of money to eradicate African swine fever. With a lot of manpower and material resources, African swine fever not only caused heavy damage to the pig industry, but also affected the veterinary medicine and feed industries. The influencing factors of my country's pig industry chain are complex. In the face of the African swine fever epidemic, in addition to improving the diagnosis level and actively developing vaccines to provid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12Q1/6806
CPCC12Q1/701C12Q1/6851C12Q1/6806C12Q2600/166C12Q2531/113C12Q2561/101C12Q2563/107C12Q2545/113C12Q2527/125C12Q2563/143Y02A50/30
Inventor 姬宏超万艳娟胡东雄阳享元
Owner 广州赛百纯生物科技有限公司
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