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

A technology for quantitative detection and source components, applied in biochemical equipment and methods, microbiological determination/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, kit and method for accurate quantitative digital PCR detection of dog-derived component
  • Primers, probe, kit and method for accurate quantitative digital PCR detection of dog-derived component
  • Primers, probe, kit and method for accurate quantitative digital PCR detection of dog-derived component

Examples

Experimental program
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Effect test

Embodiment 1

[0025] In this example, the specificity and sensitivity of the dog meat primer pair and probe are evaluated through the following tests.

[0026] In this example, the specificity of dog meat primer pairs and probes was evaluated through the following tests.

[0027] The inventors of the present invention detect the dog RPA1 gene sequence by real-time fluorescent PCR (probe method) for the first time, and can determine the specificity of the dog primer-probe combination. Reaction system: 12.5 μL of 2×TaqMan Universal PCR Master Mix; 0.5 μL of probe (10 μM); 1 μL of upstream and downstream primers (10 μM); 5 μL of template DNA; add ddH 2 O to a total volume of 25 μL. The reaction program was 95°C for 10 min; 95°C for 15 s; 60°C for 1 min, 40 cycles.

[0028] The sequences of primers and probes used to detect dog-derived components are:

[0029] The primer sequences are SEQ ID No.1 and SEQ ID No.2, the probe sequence is SEQ ID No.3, a fluorescent quenching group BHQ1 is connec...

Embodiment 2

[0057] This embodiment is to use the primer pair and probe of the dog RPA1 gene to quantitatively detect the copy number of the dog meat RPA1 gene by digital PCR and evaluate the sensitivity of the dog meat primer probe through the following test.

[0058] The inventors of the present invention detect the dog RPA1 gene sequence by digital PCR (probe method) for the first time, and can determine the specificity of the dog primer-probe combination. Reaction system: 10 μL of 2 × ddPCR Master Mix (Bio-Rad, USA); 0.5 μL of probe (10 μM); 1 μL of upstream and downstream primers (10 μM); 5 μL of template DNA; 2 O to a total volume of 20 μL. The reaction program was 95°C for 5 min (1°C / s); 94°C for 15 s, 60°C for 1 min (1°C / s), a total of 50 cycles; 98°C for 10 min (1°C / s). After the amplification, the copy number of the dog RPA1 gene was read by a droplet analyzer.

[0059] The sequences of primers and probes used to detect dog-derived components are:

[0060] The primer sequences...

Embodiment 3

[0090] This example provides a kit for the precise quantification of dog-derived components. The kit includes the specific oligonucleotide primer pair and probe used in the quantitative detection of dog-derived components by the digital PCR method of the present invention, as well as instructions for use. The kit includes a primer pair of SEQ ID No.1, SEQ ID No.2 and a probe of SEQ ID No.3, the 3' end of the probe is connected with a fluorescent quenching group BHQ1, and the 5' end is connected with a fluorescent Reporting group FAM, PCR amplification conditions are given in the instruction manual, the conditions are: 95°C, 5min (1°C / s); 94°C for 15 s, 60°C for 1 min (1°C / s) , 50 cycles in total; 98°C for 10min (1°C / s). For different instruments, the reaction parameters should be adjusted appropriately.

[0091] In order to ensure the feasibility of the established method, 3 commercially available samples were selected, including dog meat soup, spiced dog meat, dried dog mea...

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Abstract

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

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the invention relates to oligonucleotide primers and probes for the detection of dog-derived components, a digital PCR detection method for determining dog-derived components, and for accurate and quantitative detection of dog-derived components. Application of a digital PCR detection kit for dog-derived components and specific oligonucleotide primers and probes for dog-derived components in the detection of dog-derived components. 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 necessary for the human body, accounting for about 10% of the total fat content. 20%. The production and consumption of meat products across the country continue to increase. In 2013, my country became the largest me...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 黄文胜陈颖任君安吴亚君葛毅强邓婷婷
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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