Method for quantitatively detecting quail-derived components based on ddPCR technology
A technology derived from quail, applied in the field of molecular biology analysis, can solve problems such as inaccurate quantification and regulatory obstacles, and achieve the effects of convenient and fast determination process, good specificity and sensitivity, good repeatability and stability
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[0038] Example 1: Design of primer probes
[0039] Primers and probes were synthesized by Nanjing GenScript Company.
[0040] For the single-copy gene design in the quail nuclear genome, the specific probe and primer sequences are as follows:
[0041] Probe: 5'-AGCAGCCAACACAGCACCCA-3', SEQ ID NO.1;
[0042] Upstream primer: 5'-ACAGCAACGTTCTTCAGCTC-3', SEQ ID NO.2;
[0043] Downstream primer: 5'-CTGGGAGCGCTTGCTTTATT-3', SEQ ID NO.3;
[0044] The 5' end of the probe is modified with FAM, and the 3' end is modified with BHQ1; wherein, FAM represents a fluorescent reporter group, and BHQ1 represents a quencher group.
[0045] The present invention adopts the fluorescent probe method, and its detection principle is to identify the template by using fluorescently labeled specific probes. Compared with the SYBR dye method in the prior art, the specificity of the fluorescently labeled specific probe of the present invention is stronger and the background interference is lower.
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[0046]Example 2: Specificity verification of primer probes
[0047] (1) Samples: Quail meat, chicken meat, duck meat, goose meat, pigeon meat, mutton, beef and pork samples were obtained from the local farmers market in Nanjing.
[0048] (2) Reagents and instruments: DNA extraction kits were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Premix ExTaq (Takara, Japan); American Bio-Rad and CFX96 Touch fluorescence PCR instrument.
[0049] (3) Specificity verification: DNAs of quail, chicken, duck, goose, mutton, beef and pork were extracted as templates, and the primer probes in Example 1 were used for QPCR amplification, and three samples were made for each sample. Repeat, while ending with ddH 2 O instead of DNA template was used as blank control group.
[0050] Amplification reaction system: Premix Ex Taq 12.5 μL, upstream and downstream primers 0.5 μL each, probe 1 μL, template 1 μL, ddH 2 O 9.5 μL.
[0051] The reaction conditions were: 95 °C for 5 m...
Example Embodiment
[0053] Example 3: Standard curve design and linear range verification for quail-derived ddPCR quantitative detection
[0054] (1) Samples: Quail meat samples were obtained from local farmers' markets in Nanjing. Divide the quail meat sample into small pieces of about 1 cm × 1 cm × 1 cm and use the vacuum freeze-drying technology to dry the samples under the vacuum degree of 0.100 mbar and the temperature of -60 °C for 48 to 72 h to reduce the moisture content in the samples. Effects of quail-derived component concentrations. After freeze-drying, the samples are ground into powder samples by a tissue grinder to ensure the uniformity of the samples and avoid errors caused by uneven samples on the quantitative results.
[0055] (2) Reagents and instruments: DNA extraction kits were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. ddPCR amplification master mix ddPCR Super mix for Probes (Bio-Rad, USA); QX200 microdroplet digital PCR system and CFX96 Touch fluor...
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