Primer probe and method for performing real-time fluorescent polymerase chain reaction (PCR) detection on origin ingredients of raccoon

A technology of real-time fluorescence and source components, applied in the field of food inspection, to achieve the effects of improving the food inspection system, increasing protection, good sensitivity and specificity

Inactive Publication Date: 2013-12-18
中国检验认证集团吉林有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In summary, given that there is currently no detection technology that can quickly and easily detect and identify raccoon-derived componen...

Method used

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  • Primer probe and method for performing real-time fluorescent polymerase chain reaction (PCR) detection on origin ingredients of raccoon
  • Primer probe and method for performing real-time fluorescent polymerase chain reaction (PCR) detection on origin ingredients of raccoon
  • Primer probe and method for performing real-time fluorescent polymerase chain reaction (PCR) detection on origin ingredients of raccoon

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Experimental program
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Embodiment 1

[0033] 1. Primers and probes used:

[0034] Select the mitochondrial d-loop gene of raccoon dogs as the object, design and synthesize specific amplification primers and probes for real-time fluorescent PCR;

[0035] Real-time fluorescent PCR primer and probe sequences include:

[0036] Upstream primer P1: CTATACCTGGCATCTGGTTCTTACC;

[0037] Downstream primer P2: GTCCCATTTGAGTGGATTAGTAGGA;

[0038] Probe: CAGGGCCATGAACTCCCTCCATCC, the 5. end of the probe is labeled with the reporter fluorescent dye FAM, and the 3. end is labeled with the quenching fluorescent dye eclipse.

[0039] 2. The extraction and purification of sample DNA are carried out according to the following steps:

[0040] a. Use a tissue homogenizer to beat the meat sample containing raccoon-derived components into a homogenate,

[0041] Take 100 mg of homogenate and add 1.0 mL of CTAB lysate and 50 μL of proteinase K solution, vortex for 30 s, invert and mix 5 times, and place in a 65°C water bath overnight ...

Embodiment 2

[0063] Take raccoon meat, raccoon meat products, beef, pork, mutton, cat meat, dog meat, venison, camel meat, horse meat, donkey meat, rabbit meat, chicken, duck, goose, mink, fox and other 17 kinds of animal DNA In real-time fluorescent PCR detection, there is no amplification signal in samples that do not contain raccoon dog-derived components, but only raccoon dog meat has amplification signals. Example 2 shows that the primers and probes of the present invention have good specificity. see results figure 2 .

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Abstract

The invention discloses a primer and probe for performing real-time fluorescent polymerase chain reaction (PCR) detection on origin ingredients of a raccoon as well as a specific detection method. The primer probe has high sensitivity and specificity, whether the origin ingredients of the raccoon are contained in livestock meat foods can be qualitatively analyzed by adopting a real-time fluorescent PCR specific amplification method, the advantages of the real-time fluorescent PCR detection technology are exerted, a food detection system is further perfected, and the protection degree of consumer benefits is intensified.

Description

technical field : [0001] The invention belongs to the technical field of food inspection, and specifically relates to a real-time fluorescent PCR detection primer, a probe, and a method for identifying and detecting components derived from raccoon dogs using the primer probe. Background technique [0002] At present, there are two main techniques used to detect animal ingredients in food and feed at home and abroad: ELISA method and PCR specific amplification method. There are also ordinary qualitative PCR and real-time fluorescent PCR. Real-time fluorescent PCR technology is developed on the basis of ordinary PCR technology. A specific fluorescent probe is added to the amplification reaction system, and the entire PCR process is monitored in real time by using the accumulation of fluorescent signals, that is, to detect the amplification of the target nucleotide sequence. Its main advantages are: (1) Amplified product detection is performed in a closed tube state, without p...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 刘金华史艳宇刘韬孟日增聂丹丹
Owner 中国检验认证集团吉林有限公司
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