阅读说明：本技术 一种肉制品中pcr反应检测鼠源性成分的体系及方法 (The system and method for PCR reaction detection mouse ingredient in a kind of meat products ) 是由 刘慧玲 涂晓波 赵芳 万志刚 吕敬章 黄欣迪 匡燕云 于 2019-08-14 设计创作，主要内容包括：本发明涉及一种肉制品中PCR反应检测鼠源性成分的体系及方法,该肉制品中PCR反应检测鼠源性成分的方法,包括以下步骤：S1、提取待测样品DNA；S2、设计引物和荧光标记探针：以16S rRNA基因为参考,从GenBank中获取基因序列作为模板,合成引物和荧光标记探针；上游引物F的碱基序列：5’-TCCAGGTCGGTTTCTATC-3’；下游引物R的碱基序列：5’-TCTGCCACCCTAATAACC-3’；荧光标记探针序列P的碱基序列：5’-FAM-AGTACGAAAGGACA-MGB-3’；S3、建立实时荧光PCR反应体系,将步骤S1提取的待测样品DNA作为模板；将实时荧光PCR反应混合液、DNA模板、上游引物、下游引物、荧光标记探针、Rox参比染料混合并采用双蒸水补足,建立实时荧光PCR反应体系；S4、PCR扩增反应；S5、分析鼠源性成分。该方法具有操作简便、检测灵敏度高的优点。(The present invention relates to the system and method for PCR reaction detection mouse ingredient in a kind of meat products, the method for PCR reaction detection mouse ingredient in the meat products, comprising the following steps: S1, extract sample to be tested DNA；S2, design primer and fluorescence labeling probe: being reference with 16S rRNA gene, and gene order is obtained from GenBank as template, synthetic primer and fluorescence labeling probe；The base sequence of upstream primer F: 5 '-TCCAGGTCGGTTTCTATC-3 '；The base sequence of downstream primer R: 5 '-TCTGCCACCCTAATAACC-3 '；The base sequence of fluorescence labeling probe sequence P: 5 '-FAM-AGTACGAAAGGACA-MGB-3 '；S3, real-time fluorescence PCR reaction system is established, the sample to be tested DNA that step S1 is extracted is as template；Real-time fluorescence PCR reaction mixture, DNA profiling, upstream primer, downstream primer, fluorescence labeling probe, Rox reference dye are mixed and distilled water is used to supply, establishes real-time fluorescence PCR reaction system；S4, pcr amplification reaction；S5, analysis mouse ingredient.This method has the advantages that easy to operate, detection sensitivity is high.)

1. a kind of method of PCR reaction detection mouse ingredient in meat products, which comprises the following steps:

S1, sample to be tested DNA is extracted；

S2, design primer and fluorescence labeling probe: being reference with 16S rRNA gene, and gene order is obtained from GenBank and is made For template, synthetic primer and fluorescence labeling probe；

The base sequence of upstream primer F:

5'-TCCAGGTCGGTTTCTATC-3'；

The base sequence of downstream primer R:

5'-TCTGCCACCCTAATAACC-3'；

The base sequence of fluorescence labeling probe sequence P:

5'-FAM-AGTACGAAAGGACA-MGB-3'；

S3, establish real-time fluorescence PCR reaction system: the sample to be tested DNA that step S1 is extracted is as template；By real-time fluorescence PCR reaction mixture, the DNA profiling, the upstream primer, the downstream primer, the fluorescence labeling probe, Rox reference Dyestuff mixes and distilled water is used to supply, and establishes the real-time fluorescence PCR reaction system；

S4, pcr amplification reaction: progress initial denaturation is anti-under the conditions of the real-time fluorescence PCR reaction system is placed in 95 DEG C of temperature It answers；It is placed on carrying out reaction of degeneration (RD) under the conditions of 95 DEG C of temperature again, and anneals under the conditions of 60 DEG C of temperature, circuit sequentially 30~ 45 times, in 60 DEG C of collection fluorescence signals；

S5, analysis mouse ingredient: the Average Ct values of real-time fluorescence PCR reactant are measured and is calculated, and are judged in sample to be tested Whether mouse ingredient is contained；

When value≤35.0 Ct, then it is determined as that sample to be tested contains mouse ingredient；

When value >=40.0 Ct, then it is determined as sample to be tested without containing mouse ingredient；

As 35.0 < Ct value < 40.0, then repeatedly step S1~S4, Ct value is still < 40.0 after expanding again, then determines to be measured Sample contains mouse ingredient；Value >=40.0 Ct after expanding again are then determined as sample to be tested without containing mouse ingredient.

2. the method for PCR reaction detection mouse ingredient in meat products according to claim 1, which is characterized in that described The following steps are included: taking sample to be tested in centrifuge tube, addition lysate carries out centrifugation reaction and takes it after centrifugation S1 step Supernatant；Solvent is added to mix；Precipitating reagent is added to form precipitating, removes supernatant, and wash to the precipitating, dries in the air It is dry；Solvent is added and dissolves the precipitating, DNA solution is made and is saved in -20 DEG C stand-by；

Alternatively, the S1 step includes using DNA extraction kit, the genomic DNA in the tissue of sample to be tested is extracted.

3. the method for PCR reaction detection mouse ingredient in meat products according to claim 2, which is characterized in that described The concentration of DNA solution is the 10 μ g/mL of μ g/mL~100, and A260/A280 light absorption value ratio is between 1.7~1.9.

4. the method for PCR reaction detection mouse ingredient in meat products according to claim 1, which is characterized in that in institute It states in S3 step, the concentration of the upstream primer is 10 μm of ol/L.

5. the method for PCR reaction detection mouse ingredient in meat products according to claim 1, which is characterized in that in institute It states in S3 step, the concentration of the downstream primer is 10 μm of ol/L.

6. the method for PCR reaction detection mouse ingredient in meat products according to claim 1, which is characterized in that in institute It states in S3 step, the concentration of the fluorescence labeling probe is 10 μm of ol/L.

7. the method for PCR reaction detection mouse ingredient in meat products according to claim 1, which is characterized in that in institute It states in S3 step, the usage ratio of the upstream primer and the fluorescence labeling probe can be 1:1；

The downstream primer and the usage ratio of the fluorescence labeling probe can be 1:1.

8. the method for PCR reaction detection mouse ingredient in meat products according to claim 3, which is characterized in that in institute It states in S3 step, the volume ratio of each component are as follows:

Real-time fluorescence PCR reaction mixture: 50%；

Upstream primer: 4%；

Downstream primer: 4%；

Fluorescence labeling probe: 4%；

Sample to be tested DNA profiling: 4%；

Rox reference dye: 1%

Distilled water: surplus.

9. the method for PCR reaction detection mouse ingredient in meat products according to claim 1, which is characterized in that in institute It states in S4 step, by the way that the real-time fluorescence PCR reaction system is placed on fluorescent PCR instrument and sets corresponding parameter to carry out PCR amplification.

10. it is described in any item to be applied to claim 1 to 9 for the system of PCR reaction detection mouse ingredient in a kind of meat products The method of PCR reaction detection mouse ingredient in meat products, which is characterized in that including real-time fluorescence PCR reaction mixture, upstream Primer, downstream primer, fluorescence labeling probe, sample to be tested DNA profiling, Rox reference dye, distilled water；

Wherein, the base sequence of upstream primer F:

5'-TCCAGGTCGGTTTCTATC-3'；

The base sequence of downstream primer R:

5’-TCTGCCACCCTAATAACC-3’

The base sequence of fluorescence labeling probe sequence P:

5’-FAM-AGTACGAAAGGACA-MGB-3’。

Technical field

The present invention relates to animal derived materials detection technique fields, anti-more specifically to PCR in a kind of meat products The system and method for mouse ingredient should be detected.

Background technique

Currently, China generallys use this molecular biology method of PCR amplification to the detection of animal derived materials, for packet Include mouse ingredient animal derived materials detection patent and article also both for cytochrome b design primer probe, then Carry out PCR amplification.And conservative of the 16S rRNA gene in structure and function with height can be utilized due to being of moderate size Sequencing technologies relatively easily obtain its sequence, are classified scholar and are received, but the rare report for animal derived materials detection, It has no and is applied to mouse composition detection.We are according to 16S rRNA gene design primer probe in mouse cell mitochondrial, to food Mouse ingredient is detected in product, provides new approach for quickly detection mouse ingredient.

Summary of the invention

The technical problem to be solved in the present invention is that providing a kind of high sensitivity, PCR in efficiently meat products easy to detect The system and method for reaction detection mouse ingredient.

The technical solution adopted by the present invention to solve the technical problems is: constructing PCR reaction detection mouse in a kind of meat products The method of derived component, comprising the following steps:

S1, sample to be tested DNA is extracted；

S2, design primer and fluorescence labeling probe: it is reference with 16S rRNA gene, gene sequence is obtained from GenBank Column are used as template, synthetic primer and fluorescence labeling probe；

The base sequence of upstream primer F:

5'-TCCAGGTCGGTTTCTATC-3'；

The base sequence of downstream primer R:

5’-TCTGCCACCCTAATAACC-3’

The base sequence of fluorescence labeling probe sequence P:

5'-FAM-AGTACGAAAGGACA-MGB-3'；

S3, establish real-time fluorescence PCR reaction system: the sample to be tested DNA that step S1 is extracted is as template；It will be glimmering in real time Light PCR reaction mixture, the DNA profiling, the upstream primer, the downstream primer, the fluorescence labeling probe, Rox ginseng It is mixed than dyestuff and distilled water is used to supply, establish the real-time fluorescence PCR reaction system；

S4, pcr amplification reaction: the real-time fluorescence PCR reaction system is placed under the conditions of 95 DEG C of temperature and is become in advance Property reaction；It is placed on carrying out reaction of degeneration (RD) under the conditions of 95 DEG C of temperature again, and anneals under the conditions of 60 DEG C of temperature, circuit sequentially 30~45 times, in 60 DEG C of collection fluorescence signals；

S5, analysis mouse ingredient: the Average Ct values of real-time fluorescence PCR reactant are measured and is calculated, and are judged to test sample Whether contain mouse ingredient in product；

When value≤35.0 Ct, then it is determined as that sample to be tested contains mouse ingredient；

When value >=40.0 Ct, then it is determined as sample to be tested without containing mouse ingredient；

As 35.0 < Ct value < 40.0, then repeatedly step S1~S4, Ct value is still < 40.0 after expanding again, then determines Sample to be tested contains mouse ingredient；When again expand after value >=40.0 Ct, then be determined as sample to be tested without containing mouse at Point.

Preferably, lysate is added the following steps are included: take sample to be tested in centrifuge tube in the S1 step, carry out from The heart reacts and takes its supernatant after centrifugation；Solvent is added to mix；Precipitating reagent is added to form precipitating, removes supernatant, and right The precipitating is washed, and is dried；Solvent is added and dissolves the precipitating, DNA solution is made and is saved in -20 DEG C stand-by；

Alternatively, the S1 step includes using DNA extraction kit, the genomic DNA in the tissue of sample to be tested is extracted.

Preferably, the concentration of the DNA solution is the 10 μ g/mL of μ g/mL~100, and A260/A280 light absorption value ratio is 1.7 Between~1.9.

Preferably, in the S3 step, the concentration of the upstream primer is 10 μm of ol/L.

Preferably, in the S3 step, the concentration of the downstream primer is 10 μm of ol/L；

Preferably, in the S3 step, the concentration of the fluorescence labeling probe is 10 μm of ol/L.

Preferably, in the S3 step, the upstream primer and the usage ratio of the fluorescence labeling probe can be 1:1；

The downstream primer and the usage ratio of the fluorescence labeling probe can be 1:1.

Preferably, in the S3 step, the volume ratio of each component are as follows:

Real-time fluorescence PCR reaction mixture: 50%；

Upstream primer: 4%；

Downstream primer: 4%；

Fluorescence labeling probe: 4%；

Sample to be tested DNA profiling: 4%；

Rox reference dye: 1%

Distilled water: surplus.

Preferably, in the S4 step, by the way that the real-time fluorescence PCR reaction system is placed on fluorescent PCR instrument simultaneously Corresponding parameter is set to carry out PCR amplification.

The system of PCR reaction detection mouse ingredient in meat products of the present invention is applied in meat products of the present invention The method of PCR reaction detection mouse ingredient, including real-time fluorescence PCR reaction mixture, upstream primer, downstream primer, fluorescence Label probe, sample to be tested DNA profiling, Rox reference dye, distilled water；

Wherein, the base sequence of upstream primer F:

5'-TCCAGGTCGGTTTCTATC-3'；

The base sequence of downstream primer R:

5’-TCTGCCACCCTAATAACC-3’

The base sequence of fluorescence labeling probe sequence P:

5’-FAM-AGTACGAAAGGACA-MGB-3’。

Implement the Fluorescence PCR system and method for mouse ingredient in a kind of detection food of the invention, has following The utility model has the advantages that in the meat products PCR reaction detection mouse ingredient method, pass through first extract sample to be tested DNA, It is secondary, it is reference with 16S rRNA gene, the base sequence of upstream primer F is designed as 5 '-TCCAGGTCGGTTTCTATC-3 ', The base sequence of downstream primer R is designed as 5 '-TCTGCCACCCTAATAACC-3 ', by the base of fluorescence labeling probe sequence P Sequence design is 5 '-FAM-AGTACGAAAGGACA-MGB-3 '；Then, by real-time fluorescence PCR reaction mixture, Rox reference Dyestuff mixes with above-mentioned substance and distilled water is used to supply, and establishes real-time fluorescence PCR reaction system, and carry out pcr amplification reaction With mouse constituent analysis, mouse ingredient in food is detected to realize.PCR reaction detection source of mouse in the meat products Property ingredient method have the advantages that easy to operate, detection sensitivity and detection accuracy are high, at low cost and time saving and energy saving, standardize The verifying and assessment of specificity and sensitivity in mouse ingredient fluorescence PCR detection method.

The real-time fluorescence PCR reaction system of mouse ingredient, meat for use in the present invention in detection food of the invention The method of PCR reaction detection mouse ingredient in product, by real-time fluorescence PCR reaction mixture, the DNA mould of sample to be tested Plate, F base sequence be the upstream primer of 5 '-TCCAGGTCGGTTTCTATC-3 ', R base sequence be 5 '- The base sequence of the downstream primer of TCTGCCACCCTAATAACC-3 ', P are as follows: 5 '-FAM-AGTACGAAAGGACA-MGB-3's ' Fluorescence labeling probe, Rox reference dye are mixed to prepare, and have the advantages that testing result is reliable, high sensitivity.

Detailed description of the invention

Present invention will be further explained below with reference to the attached drawings and examples, in attached drawing:

Fig. 1 is the expansion of the primer and probe of the real-time fluorescence PCR reaction system of mouse ingredient in present invention detection food Increase figure；

Fig. 2 shows the flow charts of the method for PCR reaction detection mouse ingredient in meat products of the present invention；

Fig. 3 is the amplification of the method specific experiment of PCR reaction detection mouse ingredient in 1 meat products of the embodiment of the present invention Figure；

Fig. 4 is that the method for PCR reaction detection mouse ingredient in the meat products of the present invention of the embodiment of the present invention 2 detects yellow chest The amplification figure of mouse sensitivity experiment；

Fig. 5 is that the method for PCR reaction detection mouse ingredient in the meat products of the present invention of the embodiment of the present invention 2 detects small family The amplification figure of the sensitivity experiment of mouse；

Fig. 6 is that the method for PCR reaction detection mouse ingredient in the meat products of the present invention of the embodiment of the present invention 2 detects brown family The amplification figure of the sensitivity experiment of mouse；

Fig. 7 is that the method for PCR reaction detection mouse ingredient in the meat products of the present invention of the embodiment of the present invention 3 detects yellow chest The amplification figure of the repeated experiment of mouse；

Fig. 8 is that the method for PCR reaction detection mouse ingredient in the meat products of the present invention of the embodiment of the present invention 3 detects small family The amplification figure of the repeated experiment of mouse；

Fig. 9 is that the method for PCR reaction detection mouse ingredient in the meat products of the present invention of the embodiment of the present invention 3 detects brown family The amplification figure of the repeated experiment of mouse；

Figure 10 is the method detection food of PCR reaction detection mouse ingredient in the meat products of the present invention of the embodiment of the present invention 4 The amplification figure of the mouse ingredient of mouse ingredient simulation mixing meat sample in product.

Specific embodiment

For a clearer understanding of the technical characteristics, objects and effects of the present invention, now control attached drawing is described in detail A specific embodiment of the invention.

The system of PCR reaction detection mouse ingredient, can be used for detecting source of mouse in meat products in meat products of the invention Property ingredient comprising real-time fluorescence PCR reaction mixture (Premix Ex Taq (Probe qPCR)), upstream primer, downstream are drawn Object, fluorescence labeling probe, sample to be tested DNA profiling, Rox reference dye (ROX Reference Dye ∥) and distilled water.It should Real-time fluorescence PCR reaction system its by the way that said components are sufficiently mixed uniformly, and carry out brief centrifugation be made.

Wherein, real-time fluorescence PCR reaction mixture (Premix Ex Taq (Probe qPCR)) is that can pass through commercially available acquisition Conventional products, account for the 50% of the real-time fluorescence PCR reaction system total volume.

Wherein, the base sequence of upstream primer F is 5 '-TCCAGGTCGGTTTCTATC-3 ', the base sequence of downstream primer R 5 '-TCTGCCACCCTAATAACC-3 ' are classified as, the base sequence of fluorescence labeling probe sequence P is 5 '-FAM- AGTACGAAAGGACA-MGB-3'；The upstream primer and the usage ratio of the fluorescence labeling probe can be 1:1；Draw in the downstream The usage ratio of object and the fluorescence labeling probe can be 1:1；The probe of the upstream primer, downstream primer and its fluorescent marker is equal Account for the 4% of the real-time fluorescence PCR reaction system total volume.Preferably, the concentration of the upstream primer is 10 μm of ol/L, downstream primers Concentration be 10 μm of ol/L, the concentration of fluorescence labeling probe is 10 μm of ol/L.16S rRNA sequence is logical corresponding to the primer It crosses and what full-length genome comparison was found is carried out to different mouse kinds, and be currently to be used for mouse Components identification, one for the first time Level structure has opposite conservative, and secondary structure has spiral otherness again, therefore 16SrRNA is considered as that evolutionary rate compares Moderate gene can be used for detecting animal derived materials.

Fig. 1 is the foundation of the system of PCR reaction detection mouse ingredient in meat products.When the ratio of primer and probe is 1: When 1, under the premise of guaranteeing the sensitivity of detection, the ratio of 1:1 forms amplification curve earlier, while saving probe dosage, because The ratio-dependent of primer and probe is 1:1 in this Fluorescence PCR system, i.e. the final concentration of primer and probe is respectively 0.4 μ Mol/L, testing result are as shown in Figure 1.In Fig. 1,1-3: primer and probe ratio are respectively 2:1,1:1 and 2:3；4: blank pair According to.

The probe of the upstream primer, downstream primer and its fluorescent marker can search for Rattusflauipectus, house mouse, brown from NCBI The full-length genome information of home mouse obtains gene order from GenBank and goes out as stencil design, and the target gene of this experiment is root The 16s rRNA gene reference sequence included according to Genbank and design, wherein target gene is in GenBank accession number and position point It is not: Rattus norvegicus NC_001665.2,1094-2664；Rattusflauipectus NC_011638.1,1094-2659；House mouse NC_ 005089.1,1094-2675.

The DNA profiling of the sample to be tested can be extracted from sample to be tested, and concentration is 10 μ of μ g/mL~100 g/ ML, and A260/A280 light absorption value ratio is between 1.7~1.9, therefore its suitable for PCR amplification in the present embodiment can concentration It can be preferably the 10 μ g/mL of μ g/mL~60, and it accounts for the 4% of the real-time fluorescence PCR reaction system total volume.

Rox reference dye (ROX Reference Dye ∥) accounts for the 1% of the real-time fluorescence PCR reaction system total volume；It is double Steaming water can be used to supply the real-time fluorescence PCR reaction system total volume, be surplus.

It is prepared referring to the kit that TAKARA company produces, mouse ingredient is real-time in the food of every part of test sample Fluorescent PCR system (25 μ L) is as follows:

Component	Volume (μ L)
		Premix Ex Taq(Probe qPCR)(2×)	12.5
Upstream primer (10 μm of ol/L)	1.0
		Downstream primer (10 μm of ol/L)	1.0
Fluorescence labeling probe (10 μm of ol/L)	1.0
		Template DNA	1.0
ROX Reference Dye‖(50×)	0.25
		Sterilize distilled water	8.25
Total volume	25

Fig. 2 shows the methods of PCR reaction detection mouse ingredient in meat products of the present invention: the following steps are included:

S1, extraction sample to be tested DNA specifically weigh sample to be tested, and sample to be tested is taken to be placed in centrifuge tube, are added Lysate carries out centrifugation reaction；Its supernatant is taken after centrifugation reaction；Solvent is added to mix；Precipitating reagent is added to form precipitating, goes It washs, dries except supernatant, and to the precipitating；Lytic agent dissolution precipitating is added, DNA solution is made, and save in -20 DEG C For use.Wherein, the amount of lysate can be 600uL~800uL, can also be placed in water bath with thermostatic control and carry out after addition lysate Cracking reaction 30min；The solvent for taking its supernatant to be added after centrifugation reaction can be the mixed solvent of chloroform and isoamyl alcohol, Its ratio can be chloroform/isoamyl alcohol 24:1；It will of course be understood that ground is not limited to trichlorine in some other embodiment The mixed solvent of methane and isoamyl alcohol；Wherein, precipitating reagent can be the isopropanol of pre-cooling；It is to be appreciated that in some other reality It applies in example, is not limited to the isopropanol of pre-cooling；Wherein, the lytic agent for dissolving precipitating can be ethyl alcohol, it is possible to understand that ground, other one In a little embodiments, which is not limited to ethyl alcohol.

It will of course be understood that ground, can also extract box using DNA in some other embodiment and extract sample DNA；It should DNA extracts box and extracts the equivalent above-mentioned DNA extraction process to be measured of sample to be tested DNA.

S2, design primer and fluorescence labeling probe, with 16S rRNA gene be reference, the upstream primer, downstream primer and The probe of its fluorescent marker can search for the full-length genome information of Rattusflauipectus, house mouse, Rattus norvegicus, these three mouse from NCBI 16S rRNA gene order is as follows:

Rattusflauipectus:

2493

CTGAGTTCAGACCGGAGCAATCCAGGTCGGTTTCTATCTATTCACAATTTCTCCCAGTAC 2552

2553

GAAAGGACAAGAGAAATGGAGCCACCTTACC-ATAAGTGCTCCCAAACCAATTTATGAAA 2611

2612

AAAACTCAATAAAATATGTATGTACAACAAATTCACCTAGACCAGGTTATTAGGGTGGCA 2671

2672

GAGCCAGGTAATTGCGTAAGACTTAAAACCTTGTTCCCAGAGGTTCAAATCCTCTCCCTA 2731

House mouse:

2492

TAAAGTCCTACGTGATCTGAGTTCAGACCGGAGCAATCCAGGTCGGTTTCTATCTATTTA 2551

2552

CGATTTCTCCCAGTACGAAAGGACAAGAGAAATAGAGCCACCTTACAAATAAGCGCTCTC 2611

2612

AACTTAATTTATGAATAAAATCTAAATAAAATATATACGTACACCCTCTA-ACCTAGAGA 2670

2671

AGGTTATTAGGGTGGCAGAGCCAGGAAATTGCGTAAGACTTAAAACCTTGTTCCCAGAGG 2730

Rattus norvegicus:

2513

GCAATCCAGGTCGGTTTCTATCTATTTACAATTTCTCCCAGTACGAAAGGACAAGAGAAA 2572

2573

TGGAGCCTCCTTACCATAAGTGCTCCC-AACCAATTTATGAAAAAAATCTCAATAAAGTA 2631

2632

TATATGTACAATAAATTAAACCTAGCCCAGGTTATTAGGGTGGCAGAGCCAGGTAATTGC 2691

In the present embodiment, the base sequence of upstream primer F is 5 '-TCCAGGTCGGTTTCTATC-3 ', downstream is drawn The base sequence of object R is 5 '-TCTGCCACCCTAATAACC-3 ', the base sequence of fluorescence labeling probe sequence P is 5 '- FAM-AGTACGAAAGGACA-MGB-3’。

S3, real-time fluorescence PCR reaction system is established.By real-time fluorescence PCR reaction mixture (Premix Ex Taq (Probe qPCR)), upstream primer, downstream primer, fluorescence labeling probe, sample to be tested DNA profiling, Rox reference dye (ROX Reference Dye ∥) it mixes, and supplied using distilled water, and be made by centrifugation reaction.

The real-time fluorescence reaction system that step S3 is established specifically is placed on fluorescent PCR instrument by S4, pcr amplification reaction, The parameter of PCR instrument is set, which is placed under the conditions of 95 DEG C of temperature and carries out initial denaturation reaction, in advance The time of reaction of degeneration (RD) can be 20 seconds, then place it under the conditions of 95 DEG C of temperature and carry out reaction of degeneration (RD), and the time of denaturation can Think 3 seconds, certainly, denaturation time can be extended according to time situation；Real-time fluorescence reaction system after denaturation is placed in 60 DEG C temperature under the conditions of anneal, time of annealing is 30 seconds, and is circuited sequentially 30~45 times, preferably, recycles 40 times, then exists Fluorescence signal is collected under the conditions of 60 DEG C of temperature.

S5, analysis mouse ingredient specifically by using fluorescent PCR instrument, are measured and to calculate real-time fluorescence PCR anti- The Average Ct values of object are answered, and judge whether contain mouse ingredient in sample to be tested.In the present embodiment, when Ct value≤ 35.0, then it is determined as that sample to be tested contains mouse ingredient (positive)；When value >=40.0 Ct, then it is determined as that sample to be tested does not contain Mouse ingredient (feminine gender)；As 35.0 < Ct value < 40.0, then repeatedly step S1~S4, Ct value is still < after expanding again 40.0, then determine that sample to be tested contains mouse ingredient (positive)；Value >=40.0 Ct after expanding again, then be determined as to test sample Product do not contain mouse ingredient (feminine gender).

Now by specific embodiment and in conjunction with attached drawing come the side to PCR reaction detection mouse ingredient in meat products of the present invention Method does further statement.

Material and instrument

Reagent