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Method for rapidly detecting chicken, duck and pig blood components in blood jelly

A blood component, rapid technology, applied in the direction of biochemical equipment and methods, material stimulation analysis, microbial measurement/testing, etc., can solve the problems of poor repeatability, difficult quantitative analysis, and simultaneous identification of multiple species, etc., to achieve operational Convenience, improved accuracy, good economic benefits and social effects

Inactive Publication Date: 2014-01-22
NANJING JITAI BIOTECH
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  • Summary
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of target gene selection, mitochondrial DNA has a large number of copies, but it is difficult to perform quantitative analysis because of its different content in different tissues, while animal cell nuclear genomic DNA sequences are highly species-specific and can avoid non-specific amplification, making them more suitable for use. Multiplex PCR analysis; in addition, in terms of method selection, single PCR has poor repeatability and cannot simultaneously identify multiple species, while multiplex PCR can identify multiple animal-derived components at the same time, greatly improving detection efficiency and results the accuracy of
At present, there is no report on the multiplex fluorescent quantitative PCR detection method for the identification of animal blood components in xuewang, which uses the DNA sequence of the nucleus gene as the target gene in the world.

Method used

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  • Method for rapidly detecting chicken, duck and pig blood components in blood jelly
  • Method for rapidly detecting chicken, duck and pig blood components in blood jelly
  • Method for rapidly detecting chicken, duck and pig blood components in blood jelly

Examples

Experimental program
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Effect test

Embodiment 1

[0033] 1) Sample preparation and DNA extraction

[0034] Preparation of Xuewang: Add 2% salt to the collected fresh blood, wait for it to coagulate into lumps, and then boil it with boiling water.

[0035] DNA extraction: operate in accordance with the instructions of the TIANGEN (Tiangen) kit. The total DNA of 50 mg of samples (blood, donkey, pork, rabbit, goose, fish, goat, sheep, corn) was dissolved in 100 μL TE buffer, and the DNA concentration and purity were detected by a nucleic acid protein analyzer.

[0036] 2) Primer and probe design

[0037] Based on the genome sequences of duck (accession number: HQ008784.1), pig (accession number: DQ452569.1) and chicken (accession number: AY685072.1) published in GenBank, specific primers and primers were designed using Primer Premier 5.0 software. Probes, and then the primers and probe sequences were compared and evaluated by Blast analysis on the NCBI website to ensure the specificity of the primers and probes. The fluoresce...

Embodiment 2

[0049] Example 2 Analysis of the actual lower limit of detection in real-time fluorescent PCR with multiplex TaqMan probes

[0050] The DNA template stock solutions extracted from duck, pig, and chicken blood (all at a concentration of 90 ng / μL) were mixed according to 1:1:1 and then serially diluted, that is, the DNA concentrations of the three animals were respectively 30 ng / μL, 3 ng / μL, Five concentration gradients of 0.3ng / μL, 0.03ng / μL and 0.003ng / μL were used to investigate the lower limit of detection with the optimized reaction system. In order to test the linearity of the system, a standard curve was constructed for the three species, and the amplification efficiency was calculated. The formula for calculating the amplification efficiency is (E)=10 -1 / 斜率 -1, the experiment was repeated three times, and three parallels were set up each time, and the Ct value less than 36 was used as the positive criterion.

[0051] The results showed (Table 3) that when the amount of ...

Embodiment 3

[0055] Example 3 Multiplex TaqMan probe real-time fluorescent PCR sensitivity analysis

[0056] Use fresh blood to make mixed blood samples of duck blood, pig blood and chicken blood with contents of 20%, 10%, 5%, 1%, 0.1% and 0% respectively (the content is the weight content of one of the blood, The weight ratio of the remaining two kinds of blood is 1:1), and the DNA of each group was extracted, and the sensitivity of the method was analyzed according to the optimized reaction system. The experiment was carried out three times, and three parallels were set up each time.

[0057] The Ct values ​​obtained in the experiment are shown in Table 4. The results show that the system can detect 1% of duck blood, pig blood or chicken blood in Xuewang. Figure 5 , Figure 6 , Figure 7 It shows that the real-time fluorescent PCR amplification is good, and it can be confirmed that the sensitivity of this method can reach 1%, which can meet the actual detection needs.

[0058] Table ...

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Abstract

The invention discloses a method for rapidly detecting chicken, duck and pig blood components in a blood jelly. The method comprises the following steps: taking the blood jelly as a material to extract DNA (Deoxyribose Nucleic Acid); carrying out fluorescence quantitative PCR (Polymerase Chain Reaction) by using a TaqMan probe and a specific primer; applying ABI7500SofwareSDS1.4 to carry out analysis processing on an experiment result; taking amplification with a Ct value less than 36 as a positive result of the detection. The method disclosed by the invention is a multi-TaqMan probe real-time fluorescence PCR method for simultaneously detecting the chicken blood component, the duck blood component and the pig blood component in the blood jelly so as to avoid non-specific amplification; various animal original components can be identified simultaneously; the detection efficiency and the result accuracy are improved greatly; the detection lower limit is 0.15ng and the sensitivity reaches 1%; the method has good specificity and can be used for simultaneously detecting and quantifying DNA components of duck blood, pig blood and chicken blood in the blood jelly, thus exploring a new way for identifying the animal original components in food and having very good practicability.

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 rapid detection method for real-time fluorescent PCR with multiple TaqMan probes for three blood components of duck, pig and chicken in Xuewang. Background technique [0002] In recent years, food quality and safety issues, especially adulteration issues, have attracted more and more attention from society and regulatory authorities. Foods with unknown ingredients or false food labels will not only lead to unfair commercial competition among manufacturers, but may also endanger the health of consumers. Foods with unknown ingredients are potential allergens for sensitive people. In China, Japan and some European countries, animal blood has been widely used as a nutritional additive in food such as blood sausage, pudding and salad. Xuewang, also known as blood tofu, is a block formed by adding salt to a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
CPCC12Q1/686C12Q2537/143C12Q2561/101
Inventor 黄明程欣杨静黄继超周兴虎
Owner NANJING JITAI BIOTECH
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