Taqman probe fluorescent quantitation polymerase chain reaction (PCR) method for rapidly detecting pork or chicken compositions in food added with internal amplification control

A technique of amplifying internal standard and fluorescent quantification, which is applied to the rapid detection of chicken component Taqman probe fluorescent quantitative PCR and pork, to avoid false negative test results, facilitate operation, and reduce the risk of interference

Active Publication Date: 2013-01-09
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

In terms of target gene selection, animal cell nuclear genomic DNA sequences are highly species-specific, which can ensure that there will be no cross-interference caused by non-specific amplification between species during the detection process; All PCR detection studies lack positive amplification internal standard (IAC) to monitor the

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  • Taqman probe fluorescent quantitation polymerase chain reaction (PCR) method for rapidly detecting pork or chicken compositions in food added with internal amplification control
  • Taqman probe fluorescent quantitation polymerase chain reaction (PCR) method for rapidly detecting pork or chicken compositions in food added with internal amplification control
  • Taqman probe fluorescent quantitation polymerase chain reaction (PCR) method for rapidly detecting pork or chicken compositions in food added with internal amplification control

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Embodiment 1

[0024] 1. Sample preparation and DNA extraction

[0025] Cooked pork and cooked chicken are made by steaming fresh pork and fresh chicken in 100°C boiling water for 20 minutes respectively.

[0026] Weigh 50 mg of raw and cooked pork, chicken, and other animal and plant meats to extract genomic DNA according to the kit instructions, and dissolve the total DNA in 100 μL TE buffer.

[0027] Measure the absorbance value at 260nm and 280nm, and calculate the DNA concentration and purity.

[0028] 2. Primer and Probe Design

[0029] According to the published pig beta actin gene (accession number DQ452569.1) and chicken transforming growth factor gene (accession number AY685072.1) in Genbank, primers and Taqman probes were designed using Primer Express 3.0 (ABI) software, The fluorescent groups FAM and HEX were used as the luminescent groups of the probes, respectively.

[0030] The sequences of primers and probes are listed in Table 1.

[0031] Table 1 Fluorescent quantitative...

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Abstract

The invention discloses a probe fluorescent quantitation polymerase chain reaction (PCR) method for rapidly detecting pork or chicken compositions in food added with internal amplification control. The method includes designing a primer and a probe respectively based on an animal nuclear gene; and artificially synthesizing one section of competitive internal amplification control and a corresponding probe,and establishing an internal standard fluorescent quantitation PCR system respectively, using ABI 7500 Software SDS 1.4 to analyze experiment results and taking amplification with a Ct value smaller than 36 as a detection positive result. According to the method, the method is provided with good specificity aimed at target species, false negative test results are avoided by monitoring PCR reaction in real time, a novel way is explored for identification of animal origin ingredients in food, and the method has the advantages of being accurate and stable, convenient to operate and the like.

Description

technical field [0001] The invention belongs to the field of food safety detection, and relates to the rapid detection of animal-derived components in food, in particular to a Taqman probe fluorescence quantitative PCR rapid detection method for pork and chicken components in food added with amplified internal standards. Background technique [0002] The adulteration of meat products is one of the focal issues of public concern. At present, meat adulteration is mainly manifested in the use of relatively cheap pork, chicken and other meat by unscrupulous companies, through various forms of deep processing, and selling them as beef products to seek improper benefits. Adulteration not only violates the legitimate rights and interests of consumers, but also greatly undermines the public's confidence in my country's food quality and safety. Therefore, it is necessary to establish a scientific, accurate and rapid detection method for pork and chicken, which are more commonly foun...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 黄明何玮玲杨静徐幸莲周光宏
Owner NANJING AGRICULTURAL UNIVERSITY
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