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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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 repeatabilit

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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

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Example Embodiment

[0032] Example 1

[0033] 1) Sample preparation and DNA extraction

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

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

[0036] 2) Design of primers and probes

[0037] According to the published genome sequences of duck (accession number: HQ008784.1), pig (accession number DQ452569.1) and chicken (accession number AY685072.1) in GenBank, specific primers and primers were designed using Primer Premier 5.0 software. Probe, then compare and evaluate the primer and probe sequence with Blast analysis on the NCBI website to ensure the specificity of the primer and probe. The fluorescent groups FAM, CY5 and HEX were ...

Example Embodiment

[0049] Example 2 Analysis of the actual detection limit of multiple TaqMan probe real-time fluorescent PCR

[0050] The DNA template stock solution extracted from duck, pig, and chicken blood (concentrations are all 90ng / μL) was mixed 1:1:1 and then serially diluted, that is, the three animal DNA concentrations became 30ng / μL, 3ng / μL, There are 5 concentration gradients of 0.3ng / μL, 0.03ng / μL and 0.003ng / μL. The optimized reaction system is used to investigate the detection limit. In order to test the linearity of the system, a standard curve was constructed for three species, and the amplification efficiency was calculated. The amplification efficiency calculation formula is (E)=10 -1 / slope -1, the experiment was repeated three times, and three parallels were set each time, with a Ct value less than 36 as the positive criterion.

[0051] The results show (Table 3) that when the template amount of duck, pig, and chicken in the fluorescent quantitative PCR system is 0.15ng (0.03ng...

Example Embodiment

[0055] Example 3 Sensitivity analysis of multiple TaqMan probes real-time fluorescent PCR

[0056] Fresh blood was used to prepare mixed blood samples containing 20%, 10%, 5%, 1%, 0.1% and 0% of duck blood, pig blood and chicken blood (the content is the weight content of one of the blood, The weight ratio of the remaining two types of blood is 1:1), extract each group of DNA, and analyze the sensitivity of the method according to the optimized reaction system. The experiment is carried out in three stages, with three parallels each time.

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

[0058] Table 4 Sensitivity analysis results of multiple TaqMan...

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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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