Specific amplification primer pair and fluorescent quantitative PCR kit

A technology for amplification primers and fluorescent quantification, applied in the field of gene editing, can solve the problems of increasing false positives and being easily affected by external factors

Pending Publication Date: 2021-04-20
FOSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Disadvantages: The kit is easily affected by external facto

Method used

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  • Specific amplification primer pair and fluorescent quantitative PCR kit
  • Specific amplification primer pair and fluorescent quantitative PCR kit
  • Specific amplification primer pair and fluorescent quantitative PCR kit

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Experimental program
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Effect test

Embodiment 1

[0017] Embodiment 1, design and synthesis of specific amplification primer pair

[0018] The GPCR gene was amplified, and analyzed and compared according to the LSDV GPCR gene sequence (accession number: MN508357) published in GenBank. A pair of specific primers were designed, the upstream primer qGPCR-F: 5'-AGTCGAATATAAAGTAATCAGTC-3', the downstream primer qGPCR-R: 5'-CCGCATATAATACAACTTATTATAG-3', and the length of the amplified fragment was 125bp. The above primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

Embodiment 2

[0019] Embodiment 2, the construction of positive standard

[0020] The DNA of LSDV was extracted according to the Mini BEST ViralRNA / DNA Extraction Kit, and its GPCR gene was amplified by ordinary PCR. The reaction system is 25 μL: 2×Premix Tap 12.5 μL, upstream and downstream primers 1.0 μL (10 μmol / L), template 1.0 μL, ddH 2 O 7.5 μL. The amplification program was 94°C for 5min; 94°C for 30s, 55°C for 30s, 72°C for 1min, 35 cycles; 72°C for 5min. The PCR amplification products were subjected to 1.0% agarose electrophoresis, such as figure 1 shown. Then follow the instructions of DNA Purification Kit to recover and purify the PCR product, and clone it into the pMD19-T vector to construct the recombinant plasmid pMD19T-LSDV, which is the positive standard. figure 1 Middle lane number 1 is the recombinant plasmid pMD19T-GPCR, and lane number 2 is the negative control.

[0021] The recombinant plasmid pMD19T-LSDV was sent to Shanghai Sangon Bioengineering Co., Ltd. for seq...

Embodiment 3

[0022] Embodiment 3, the establishment of fluorescent quantitative PCR standard curve

[0023] The recombinant plasmid pMD19T-LSDV was serially diluted to obtain 7.11×10 2 copies / μL~7.11×10 9 Copies / μL and other 8 dilution standards are used as the reaction template. Three samples were replicated for each dilution gradient and a negative control was established. Fluorescence quantitative PCR reaction system is 20 μL: TB Green (2×) 10 μL, upstream and downstream primers 0.8 μL (10 μmol / L), template 1 μL, ddH 2 O 7.4 μL. The reaction conditions were: 95°C for 30s, 1 cycle; 95°C for 10s; 60°C for 30s, a total of 40 cycles. Take the logarithm of the standard substance concentration as the X axis, and the Cq value as the Y axis, and draw a standard curve such as figure 2 As shown, the standard curve is y=-3.1852x+37.764, and the correlation coefficient R 2 =0.9993, the amplification efficiency Efficiency=106.04%.

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Abstract

The invention discloses an upstream primer and downstream primer for specifically amplifying a GPCR gene of a lumpy skin disease virus. The invention also discloses a fluorescent quantitative PCR kit containing a specific amplification primer pair. A fluorescent dye method is adopted, the specificity is good, no amplification signal is generated for a GTPV vaccine strain, and 7.11 * 10 <2> copies/[mu]L of LSDV DNA can be detected. The method is economical and practical, can be used for drawing a dissolution curve and analyzing the Tm value of all PCR products, and is not easily influenced by external factors.

Description

technical field [0001] The disclosure belongs to the technical field of gene editing, and in particular relates to a pair of specific amplification primers and a fluorescent quantitative PCR kit containing the primers. Background technique [0002] Bovine nodular skin disease (Lumpy skin disease, LSD) is a poxviridae (Poxviridae), spinal animal poxvirus subfamily (Chordopoxvirinae), capripoxvirus (Capripoxvirus) double-stranded DNA virus (LSDV) caused by bovine viral disease. The entire genome of LSDV is about 150kb long. Like other poxviruses, the viral genome consists of a core region in the middle and inverted repeats at both ends. The clinical manifestations of affected cattle are fever, hard nodules or ulcers on the skin surface, accompanied by salivation, lacrimation and nasal discharge in the course of the disease; listlessness, weight loss and secondary bacterial infection; reduced milk production; death in severe cases . LSD is listed as a disease that must be no...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
Inventor 原耀贤孙铭澮马骏刘全马春全
Owner FOSHAN UNIVERSITY
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