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Detection method for foot and mouth disease virus in aerosol and kit

A foot-and-mouth disease virus and aerosol technology, which is applied in the field of kits, specific primers, and typing to detect foot-and-mouth disease virus in aerosols, and can solve problems such as typing methods that have not been reported.

Active Publication Date: 2014-08-13
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the detection and typing methods of FMD virus in aerosol by fluorescent quantitative PCR

Method used

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  • Detection method for foot and mouth disease virus in aerosol and kit
  • Detection method for foot and mouth disease virus in aerosol and kit
  • Detection method for foot and mouth disease virus in aerosol and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The preparation of embodiment 1 standard product positive plasmid

[0054] 1. Design and synthesis of primers

[0055] Through sequence comparison of the 5'UTR gene of foot-and-mouth disease virus in GeneBank, the conserved region was determined, and a pair of universal primers were designed, which were SEQ ID NO.1 and SEQ ID NO.2 in the sequence table, which could be used for seven serotypes of foot-and-mouth disease virus Detect and amplify the PCR fragments for the construction of positive standard plasmids; then design 3 pairs of primers according to the sequence difference of FMD virus VP1, which can be used to type O, A and Asia1 serotypes of FMD virus. The primers for typing O-type foot-and-mouth disease virus are SEQ ID NO.3 and SEQ ID NO.4 in the sequence listing; the primers for typing A-type foot-and-mouth disease virus are the sequences SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing; Asia1 The primers for typing foot-and-mouth disease virus are respect...

Embodiment 2

[0063] The optimization of the SYBR Green I fluorescent quantitative RT-PCR reaction system and condition of embodiment 2 foot-and-mouth disease virus general primers

[0064] 1. Optimization of the amount of AMV reverse transcriptase

[0065] After comparing the experimental results using different concentrations of AMV reverse transcriptase, 5U was selected as the optimal amount of AMV in the reaction system.

[0066] 2. Optimization of primer concentration

[0067] In the reaction system, the concentration of the primers were serially diluted from 0.1 μmol / L to 1.6 μmol / L, and combined with each other, they were passed through a fluorescent quantitative PCR instrument ( 480II) detection, through the analysis and comparison of the test results, the final concentration of the optimal primer determined is 0.3 μmol / L. For the FMD virus typing compound SYBRGreenI fluorescent quantitative RT-PCR reaction system, the best primer final concentration of primer 3, primer 4, primer...

Embodiment 3

[0070] The establishment of the SYBR Green I fluorescent quantitative RT-PCR detection method of embodiment 3 foot-and-mouth disease virus general primers

[0071] 1. Determination of standard curve and analysis of melting curve

[0072] Determine the standard curve of fluorescent quantitative PCR, quantify the plasmid pEAST-T3-5'UTR, and then dilute it to 3×10 by 10 times 0 -3×10 9 Copy number / μL, use the diluted copy number as a template for SYBR Green I real-time fluorescent quantitative PCR amplification, and establish a standard curve. The amplification reaction system is 20 μL: 10 μL of 2×SYBR Premix Ex Taq II, 0.5 μL of 5’UTR-F and 5’UTR-R at a concentration of 10 μmol / L, 1 μL of positive plasmid pEAST-T3-5’UTR, RNase Free ddH 2 O to make up 20 μL. At the same time, a negative control was set (replacing the template with an equal amount of pEAST-T3). Then carry out the amplification of the standard curve. The reaction conditions are pre-denaturation at 95°C for 30s,...

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Abstract

The invention discloses a detection method for the foot and mouth disease virus in an aerosol. The method comprises the following steps: (1) acquisition of an aerosol sample; (2) extraction of cDNA of the virus in the aerosol sample; (3) detection: a step of carrying out PCR amplification; (4) establishment of a standard curve and a melting curve: a step of establishing the standard curve of positive standard plasmid and the melting curve of an amplification system; and (5) judgment: a step of judging whether the aerosol sample contains the foot and mouth disease virus. The invention further discloses specific primers (as shown in SEQ ID No. 1 to 8) and a kit (composed of the specific primers, RT reaction buffer, AMV reverse transcriptase, Premix Ex Taq<TM> II with a registered trademark of SYBR, the standard positive plasmid and RNase-free water). The method and the kit provided by the invention are applicable to detection and serotyping of the foot and mouth disease virus in the aerosol in a culturing farm and to direct detection of clinical samples and can also be used for non-disease diagnosis like scientific research.

Description

technical field [0001] The invention relates to a method for typing and detecting foot-and-mouth disease virus in aerosol by the fluorescent quantitative SYBR Green I method, as well as specific primers and a kit, belonging to the field of biological detection. Background technique [0002] Foot-and-mouth disease is an acute, febrile, and highly contagious infectious disease caused by the foot-and-mouth disease virus in cloven-hoofed animals such as pigs, cattle, and sheep. Once an outbreak occurs, the infected and contact infected animals must be culled. Foot-and-mouth disease is listed as a statutory reportable disease by the World Organization for Animal Health, and is listed as the first class of animal diseases in my country. It will not only cause huge direct economic losses, but also seriously endanger the healthy development of animal husbandry and the foreign trade of related products. Politics and economics have far-reaching effects. [0003] Foot-and-mouth disease...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/70C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 王洪梅何洪彬
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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