Multiplex PCR detection kit for rapidly detecting meat-derived foods

Abstract

The invention provides a multiplex PCR detection kit for rapidly detecting meat-derived foods. The multiplex PCR detection kit is mediated by a 'public primer', can identify mouse, fox, duck and sheepcomponents in foods or animal products, and belongs to the technical field of molecular detection of animal-derived components. The kit provided by the invention discloses specific primer pairs of four species mouse, fox, duck and sheep, wherein a sheep-specific primer is a primer pair capable of specifically amplifying a nucleotide sequence shown in SEQ ID No. 1; the primer of the invention canbe adopted to amplify a sample DNA; a PCR amplification product is analyzed to determine whether the sample contains sheep-derived components; moreover, whether mouse-derived components, fox-derived components and duck-derived components are doped can be identified; in addition, the mouse-derived components, the fox-derived components, the duck-derived components and sheep-derived components can be identified at one time by multiplex PCR detection. The kit provided by the invention has relatively high sensitivity, reproducibility, simple operation, relatively low cost and wide applicability, and can be used for screening and identification detection of meat sources of mixed meat products.

Description

technical field

[0001] The invention belongs to the field of gene detection, and in particular relates to a multiplex PCR detection kit for rapid detection of meat-derived food, which can be used for identification of mutton components and rat, fox, duck and sheep components in animal products. Background technique

[0002] Mutton is not only delicious, but also nourishes blood and Qi, warms the heart and warms the kidneys. It is a good tonic product. However, due to the long growth cycle of sheep, low multiple birth rate, and high breeding costs, the price of mutton is much higher than that of chicken, duck and other meat. , and as consumers realize that mutton has higher nutritional value and the level of consumption increases, the market demand for mutton is increasing day by day. In the case of high prices and short supply, many merchants took risks and joined the "army of mutton counterfeiting". There are three main types of mutton adulteration in the market: mutton mix...

Examples

Embodiment 1

[0090] Example 1 The specificity detection of mouse, fox, duck, sheep primer pair

[0091] (1) Collect 1g each of mouse, fox, duck, and sheep samples, and store them at -20°C for later use;

[0092] (2) DNA template preparation, DNA template preparation using column animal mitochondrial DNA extraction kit PCR grade (Beijing Biolab) or other recognized extraction methods with the same efficacy; purified DNA was measured by UV spectrophotometer The obtained OD260 / OD280 are both between 1.8-2.0, and the concentration is diluted to 20ng / μL;

[0093] (3) PCR detection and result analysis

[0094] single primer single template method

[0095] The reaction reagents (as shown in Table 1) were sequentially added into the PCR reaction tube, and the primers were specific primer pairs for mouse, fox, duck, and sheep, and their corresponding templates. Carry out the PCR reaction, the procedure is shown in the appendix figure 1 ; After the reaction, the PCR tube was taken out, and the P...

Embodiment 2

[0101]Example 2 Specific detection of multiplex PCR system

[0102] (1) Collect 1g each of mouse, fox, duck, and sheep samples, and store them at -20°C for later use;

[0103] (2) DNA template preparation, DNA template preparation respectively with column type animal mitochondrial DNA extraction kit PCR grade (Beijing Biolab) or other recognized extraction methods with the same efficacy; purified DNA was detected by UV spectrophotometer The obtained OD260 / OD280 are both between 1.8-2.0, and the concentration is diluted to 20ng / μL;

[0104] (3) PCR detection and result analysis

[0105] The final concentration ratio of the primers in the kit according to the public primer, sheep upstream primer, mouse upstream primer, fox upstream primer, duck upstream primer, sheep downstream primer, mouse downstream primer, fox downstream primer, and duck downstream primer is 1.5:1:1 : 1:1:1:1:0.125:0.03 ratio mixing; firstly prepare 5 μM common primer solution, respectively configure 10 μM...

Embodiment 3

[0116] Example 3 Sensitivity detection of multiplex system

[0117] (1) Collect 1g each of mouse, fox, duck, and sheep samples, and store them at -20°C for later use;

[0118] (2) DNA template preparation, DNA template preparation using column animal mitochondrial DNA extraction kit PCR grade (Beijing Biolab) or other recognized extraction methods with the same efficacy, the purified DNA was measured by UV spectrophotometer The obtained OD260 / OD280 are both between 1.8-2.0, and the concentration is diluted to 20ng / μL;

[0119] (3) PCR detection and result analysis

[0120] The sensitivity test of the system adopts the method of multiple primers and single template. As in Example 2, the primers in the kit are divided into public primers, sheep upstream primers, mouse upstream primers, fox upstream primers, duck upstream primers, sheep downstream primers, and mouse downstream primers. The molar ratio of primers, fox downstream primers and duck downstream primers is 1.5:1:1:1:1...

technical field

[0001] The invention belongs to the field of gene detection, and in particular relates to a multiplex PCR detection kit for rapid detection of meat-derived food, which can be used for identification of mutton components and rat, fox, duck and sheep components in animal products. Background technique

[0002] Mutton is not only delicious, but also nourishes blood and Qi, warms the heart and warms the kidneys. It is a good tonic product. However, due to the long growth cycle of sheep, low multiple birth rate, and high breeding costs, the price of mutton is much higher than that of chicken, duck and other meat. , and as consumers realize that mutton has higher nutritional value and the level of consumption increases, the market demand for mutton is increasing day by day. In the case of high prices and short supply, many merchants took risks and joined the "army of mutton counterfeiting". There are three main types of mutton adulteration in the market: mutton mix...

Examples

Embodiment 1

[0090] Example 1 The specificity detection of mouse, fox, duck, sheep primer pair

[0091] (1) Collect 1g each of mouse, fox, duck, and sheep samples, and store them at -20°C for later use;

[0092] (2) DNA template preparation, DNA template preparation using column animal mitochondrial DNA extraction kit PCR grade (Beijing Biolab) or other recognized extraction methods with the same efficacy; purified DNA was measured by UV spectrophotometer The obtained OD260 / OD280 are both between 1.8-2.0, and the concentration is diluted to 20ng / μL;

[0093] (3) PCR detection and result analysis

[0094] single primer single template method

[0095] The reaction reagents (as shown in Table 1) were sequentially added into the PCR reaction tube, and the primers were specific primer pairs for mouse, fox, duck, and sheep, and their corresponding templates. Carry out the PCR reaction, the procedure is shown in the appendix figure 1 ; After the reaction, the PCR tube was taken out, and the P...

Embodiment 2

[0101]Example 2 Specific detection of multiplex PCR system

[0102] (1) Collect 1g each of mouse, fox, duck, and sheep samples, and store them at -20°C for later use;

[0103] (2) DNA template preparation, DNA template preparation respectively with column type animal mitochondrial DNA extraction kit PCR grade (Beijing Biolab) or other recognized extraction methods with the same efficacy; purified DNA was detected by UV spectrophotometer The obtained OD260 / OD280 are both between 1.8-2.0, and the concentration is diluted to 20ng / μL;

[0104] (3) PCR detection and result analysis

[0105] The final concentration ratio of the primers in the kit according to the public primer, sheep upstream primer, mouse upstream primer, fox upstream primer, duck upstream primer, sheep downstream primer, mouse downstream primer, fox downstream primer, and duck downstream primer is 1.5:1:1 : 1:1:1:1:0.125:0.03 ratio mixing; firstly prepare 5 μM common primer solution, respectively configure 10 μM...

Embodiment 3

[0116] Example 3 Sensitivity detection of multiplex system

[0117] (1) Collect 1g each of mouse, fox, duck, and sheep samples, and store them at -20°C for later use;

[0118] (2) DNA template preparation, DNA template preparation using column animal mitochondrial DNA extraction kit PCR grade (Beijing Biolab) or other recognized extraction methods with the same efficacy, the purified DNA was measured by UV spectrophotometer The obtained OD260 / OD280 are both between 1.8-2.0, and the concentration is diluted to 20ng / μL;

[0119] (3) PCR detection and result analysis

[0120] The sensitivity test of the system adopts the method of multiple primers and single template. As in Example 2, the primers in the kit are divided into public primers, sheep upstream primers, mouse upstream primers, fox upstream primers, duck upstream primers, sheep downstream primers, and mouse downstream primers. The molar ratio of primers, fox downstream primers and duck downstream primers is 1.5:1:1:1:1...

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