Primers, probe, method and kit for accurate quantitative digital PCR detection of fox-derived component

A source component, quantitative detection technology, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the deviation of quantitative results of amplification efficiency, the accuracy error of template copy number, and improve detection Cost and other issues, to achieve stable and reliable results, reliable results, accurate and sensitive results

Inactive Publication Date: 2017-12-19
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are the following problems in the quantitative detection of meat components using real-time fluorescent PCR technology: (1) Each detection operation requires the amplification of a known concentration standard and the preparation of a standard curve at the same time, which increases the detection cost and excessive workload (2) The difference between the PCR amplification efficiency of the standard product and the actual sample may cause deviations in the quantitative results; (3) There is a certain error in the accuracy of the template copy number obtained through the standard curve

Method used

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  • Primers, probe, method and kit for accurate quantitative digital PCR detection of fox-derived component
  • Primers, probe, method and kit for accurate quantitative digital PCR detection of fox-derived component
  • Primers, probe, method and kit for accurate quantitative digital PCR detection of fox-derived component

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] In this example, the fox primer pair and probe specificity were evaluated by the following test.

[0026] The inventor of the present invention detects the fox F2 gene sequence by real-time fluorescent PCR (probe method) for the first time, and can determine the specificity of the fox primer probe combination. The reaction system is: 2×TaqMan Universal PCR Master Mix 12.5 μL; probe (10 μM) 0.5 μL; upstream and downstream primers (10 μM) each 1 μL; template DNA 5 μL; add ddH 2 O to a total volume of 25 μL. The reaction program is 95°C 10 min; 95°C 15 s; 60°C 1 min, 40 cycles.

[0027] The primers and probe sequences used to detect fox-derived components are:

[0028] The primer sequence is SEQ ID No.1 and SEQ ID No.2, the probe sequence is SEQ ID No.3, a fluorescence quenching group BHQ1 is attached to the 3'end of the probe, and a fluorescence is attached to the 5'end Report group FAM.

[0029] The main testing instruments used:

[0030] Micropipette (10 µL, 100 µL, 1000 µL, E...

Embodiment 2

[0056] In this example, the fox F2 gene primer pair and probe were used in the following experiment to quantitatively detect the copy number of the fox gene and evaluate the sensitivity of the fox primer probe by digital PCR.

[0057] The inventor of the present invention detects the fox F2 gene sequence through digital PCR (probe method) for the first time, and can determine the specificity of the fox primer probe combination. The reaction system is: 2 × ddPCR Master Mix (Bio-Rad, USA) 10 μL; probe (10 μM) 0.5 μL; upstream and downstream primers (10 μM) each 1 μL; template DNA 5 μL; add ddH 2 O to a total volume of 20 μL. The reaction program is 95°C, 5 min (1°C / s); 94°C 15 s, 60°C 1 min (1°C / s), 50 cycles; 98°C 10 min (1°C / s). After the amplification, the copy number of fox F2 gene was read by a droplet analyzer.

[0058] The primers and probe sequences used to detect fox-derived components are:

[0059] The primer sequence is SEQ ID No.1 and SEQ ID No.2, the probe sequence is SE...

Embodiment 3

[0089] This embodiment provides a kit for accurately quantifying gene copy numbers of fox-derived components. The kit includes specific oligonucleotide primer pairs and probes for quantitative detection of fox-derived components in the digital PCR method of the present invention, and instructions for use. The kit includes primer pair SEQ ID No. 1, SEQ ID No. 2 and probe SEQ ID No. 3, the 3'end of the probe is connected with a fluorescence quenching group BHQ1, and the 5'end is connected with a fluorescent Reporter group FAM, the PCR amplification conditions are given in the instruction manual, and the conditions are 95℃, 5min (1℃ / s); 94℃ 15 s, 60℃ 1 min (1℃ / s) , A total of 50 cycles; 98°C for 10 min (1°C / s). For different instruments, the reaction parameters should be adjusted appropriately.

[0090] In order to ensure the feasibility of the established method, four commercially available samples were selected, including mutton rolls, donkey slices, meat sausages, beef rolls, e...

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Abstract

The invention relates to oligonucleotide primers and a probe for accurate and quantitative detection of a fox-derived component. The invention also relates to a digital PCR detection method for quantitative detection of the fox-derived component. The method comprises a step of utilizing the specific oligonucleotide primers and probe directed at the single-copy nuclear gene of the fox-derived component. The invention also relates to a digital PCR detection kit for accurate and quantitative detection of the fox-derived component. The kit comprises the specific oligonucleotide primers and probe for digital PCR detection of the fox-derived component. The invention further relates to application of the specific oligonucleotide primers and probe directed at the single-copy housekeeping gene of the fox-derived component to accurate and quantitative detection of the gene copy number of the fox-derived component and application of the specific oligonucleotide primers and probe directed at the single-copy housekeeping gene of the fox-derived component to accurate and quantitative detection of the absolute mass fraction of the fox-derived component. With the digital PCR quantitative detection method and kit of the invention, whether fresh meat and preliminarily processed meat products contain the fox-derived component or not can be specifically, sensitively and accurately detected.

Description

Technical field [0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to oligonucleotide primers and probes used for the quantitative detection of fox-derived components, a digital PCR detection method for determining fox-derived components, and is used for accurate quantification of foxes. The application of the digital PCR detection kit for the original components and the specific oligonucleotide primers and probes of the fox-derived components in the detection of the fox-derived components in the samples. Background technique [0002] Meat and its products are rich in nutrients, with a protein content of about 10%-20%. Livestock meat is rich in B vitamins. Poultry meat has a high content of unsaturated fatty acids, such as linoleic acid, which is essential for the human body, which accounts for about the total fat content. 20%. The production and consumption of meat products across the country continue to increase. In ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2531/113C12Q2563/107
Inventor 陈颖任君安黄文胜邓婷婷吴亚君葛毅强
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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