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Fluorogenic quantitative PCR primer, probe combination, kit and detecting method for fast identifying camel source ingredients

A source component, fluorescent quantitative technology, applied in the field of food inspection and molecular biology detection, to achieve the effect of good accuracy, simple operation and high sensitivity

Pending Publication Date: 2016-07-06
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no publication or report on a method for quickly identifying camel-derived species using TaqMan probe primer mix, kit and fluorescent quantitative PCR detection method

Method used

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  • Fluorogenic quantitative PCR primer, probe combination, kit and detecting method for fast identifying camel source ingredients
  • Fluorogenic quantitative PCR primer, probe combination, kit and detecting method for fast identifying camel source ingredients
  • Fluorogenic quantitative PCR primer, probe combination, kit and detecting method for fast identifying camel source ingredients

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Obtaining of the target gene of embodiment 1

[0049] The method steps for detecting the animal origin of meat products using the kit of the present invention and the real-time fluorescent quantitative PCR detection method are as follows, and can detect whether various meat samples to be tested contain camel-derived components:

[0050] DNA extraction: Take 50g of the sample to be tested, grind it and mix it thoroughly. The sample to be tested can be animal skin, hair, organ tissue and muscle. Take 50mg to extract DNA. Animal tissue extraction kits can be used to extract DNA, and classic hand-held methods can also be used (see Molecular Cloning Handbook Animal Tissue DNA Extraction Methods). The Nanodrop nucleic acid detector detects the concentration and purity of the DNA sample, which requires 1-20ng / μl, and the OD value is between 1.7-2.0.

[0051] The acquisition of the camel target gene: using the extracted camel-derived genome as a template, carry out ordinary PC...

Embodiment 2

[0052] The real-time fluorescent PCR amplification of embodiment 2 test sample DNA

[0053] This kit includes: PCR buffer, MgCl 2 Reaction mixture with dNTPs (2×TaqManMasterMix), ultrapure water, camel primers for highly specific amplification, TaqMan probe mixture (0.4~1μM)

[0054] Prepare in the PCR reaction tube according to the reaction system of the kit in Table 1, put the PCR reaction tube into the fluorescent quantitative PCR instrument, and complete the PCR amplification according to the following reaction conditions: Amplification program: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 10 s , annealing at 60° C. for 35 s (where fluorescence signals are collected), 40 cycles.

[0055] For the real-time fluorescent quantitative PCR amplification reaction system of the present invention, when it is a 20 μL reaction system, its preferred configuration is shown in Table 2.

[0056] Table 2 Fluorescent quantitative PCR reaction system

[0057]

[0058]...

Embodiment 4

[0068] The sensitivity test of embodiment 4 methodological verification

[0069] Genomic DNA Sensitivity Test

[0070] According to the kit used in Example 2, the target genomic DNA was extracted, quantified to 5ng by Nanodrop, and diluted to 0.5ng / μL, 0.05ng / μL, 0.005ng / μL, 0.0005ng / μL, 0.00005ng / μL, , 2 μl was taken as the template amount for each gradient, and the camel DNA was detected according to the above method to investigate the sensitivity of the kit.

[0071] The results show( Figure 4 ), when the amount of fluorescent quantitative template DNA is 0.001ng, the probe still has an amplification curve and the Ct value is <35; but when the template amount is 0.0001ng, the Ct value is more than 35, and there is basically no amplification curve, so the kit’s The detection limit was 0.001ng.

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Abstract

The invention provides a fluorogenic quantitative PCR specific primer, a probe and a kit of the primer and relates to the technical field of food check and molecular biological detection. The primer, the probe combination, real-time fluorescence PCR and the detection kit are utilized and combined, and therefore the camel source ingredients contained in meat products can be identified fast. The real-time fluorescence quantitative PCR technology and the fluorescence probe nucleic acid DNA molecular marker technology are applied, the camel source specific primer and the specific probe are designed according to the mitochondrion cytochrome b (Cytb) gene height keeping area, and the primer has the advantages of being high in sensitivity, good in accuracy, fast, small in degradation degree (mtDNA is kept completely in the processing process), stable and easy to operate.

Description

technical field [0001] The invention relates to the technical field of food inspection and molecular biology detection, in particular to a fluorescent quantitative PCR specific primer, probe and kit for rapidly identifying camel-derived ingredients, especially for the detection of camel-derived ingredients in meat products and identification. Background technique [0002] my country's camels are mostly produced in Inner Mongolia, Xinjiang, Gansu, Qinghai, Ningxia and other places, and are one of the largest producing areas in the world. The whole body of a camel is full of treasures, and the development and utilization of camel meat, camel milk, and camel hair are of great value, and it is a good project for farmers and herdsmen to make a fortune. Camel meat contains protein, fat, calcium, phosphorus, iron, vitamin A, vitamin B1, vitamin B2 and niacin. The meat is tender and palatable, and contains a lot of glycogen, so the meat is sweet, much like horse meat, comparable t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2531/113C12Q2561/101
Inventor 张全芳刘艳艳范阳阳步迅
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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