CN112111579A - Identification method of Tan sheep derived components - Google Patents
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Abstract
The invention discloses a method for identifying Tan sheep derived components. The method is used for identifying and distinguishing Tan sheep from other sheep species based on 35 mitochondrial SNP locus combinations or 14 core mitochondrial SNP locus combinations. The 14 Tan sheep mitochondrial SNP loci with high classification efficiency are obtained by sheep interspecies mitochondrial DNA sequence comparison and intelligent algorithm screening, and can effectively distinguish Tan sheep from non-Tan sheep. The multiplex RT-PCR melting curve method can synchronously identify the genotypes of the 14 sites of the sample to be detected, thereby completing the identification of the truth of the sample Tan sheep or Tan sheep meat. The method is quick and simple to operate, intuitive and reliable in result and low in cost, and has important significance for preventing adulteration and counterfeiting, improving the overall brand value of the Tan sheep industry and promoting healthy and rapid development of the industry.
Description
Technical Field
The invention belongs to the technical field of molecular biology and food detection, and particularly relates to a method for identifying Tan sheep derived components.
Background
The Tan sheep is a local unique fine breed which is bred by natural selection and artificial selection for a long time in China, the Tan sheep meat is tender in meat quality, rich in nutrition, free of mutton smell and greasy, is an extremely rare sheep variety for fur in the world, and has high economic value. Tan sheep has entered the list of the first national-level livestock genetic resource gene library in China.
The adulteration of meat and meat products in the market is a main problem in food processing, circulation and catering, the benefit brought by price difference drives illegal vendors and enterprises to be good, and the high-price meat products such as Tan mutton are adulterated with non-Tan mutton. Therefore, the method for identifying Tan sheep and non-Tan sheep with rapidness, effectiveness and low cost is established, a guarantee basis can be provided for identifying the truth of Tan sheep, and the method has important significance for improving the whole brand value of Tan sheep industry and protecting precious sheep species and breeding and processing enterprises thereof.
Disclosure of Invention
The invention aims to provide a method for identifying Tan sheep derived components.
Another purpose of the invention is to provide a SNP marker combination for identifying Tan sheep and application thereof.
In order to achieve the purpose of the invention, in a first aspect, the invention provides a core SNP marker combination for identifying Tan sheep, wherein the SNP marker combination comprises 14 SNP sites including SNP 1-SNP 14, and the physical position of each SNP site is determined by referring to Tan sheep mitochondrial genome sequence GenBank: KF938336.1 as follows: the SNP1 is located on the 932 th bp of mitochondria, the polymorphism is A/G, Tan sheep is A, and non-Tan sheep is A or G; SNP2 is located at 955bp on mitochondria, polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP3 is located on the 7301bp of mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP4 is located on the 7350bp of mitochondria, the polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP5 is located on the upper 12645bp of mitochondria, the polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP6 is positioned on the 12813bp of mitochondria, the polymorphism is G/A, Tan sheep is G, and non-Tan sheep is A or G; the SNP7 is located at 14873bp on mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP8 is located at 14951bp on mitochondria, the polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP9 is located at 10954bp on mitochondria, the polymorphism is C/T, Tan sheep is C, non-Tan sheep is C or T, the SNP10 is located at 11031bp on mitochondria, the polymorphism is C/T, Tan sheep is C, non-Tan sheep is C or T; the SNP11 is located on the 1371bp of mitochondria, the polymorphism is A/G, Tan sheep is A, and non-Tan sheep is A or G; the SNP12 is located at the 1512bp on mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP13 is located on the 8712bp of mitochondria, the polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP14 is located at the 8866bp on mitochondria, the polymorphism is G/A, Tan sheep is G, and non-Tan sheep is A or G.
The Tan sheep is judged when the 14 sites of SNP 1-SNP 14 are all Tan sheep genotypes, the Tan sheep is judged when at least one of the 14 sites is a non-Tan sheep genotype, and the judgment accuracy rate is larger than or equal to 82% theoretically according to the statistical calculation result of the modeling sample.
In a second aspect, the present invention provides a combination of SNP markers for identifying Tan sheep, comprising 14 core SNP markers and 21 extension SNP markers; wherein, the 14 core SNP markers comprise 14 SNP sites of the SNPs 1-14; the 21 extended SNP markers include 21 SNP sites from SNP15 to SNP 35.
For SNPs 15-35, the physical position of each SNP site is determined by referring to Tan sheep mitochondrial genome sequence GenBank KF938336.1 as follows: the SNP15 is located on the 3037bp of mitochondria, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; the SNP16 is positioned on the mitochondria at 3299bp, the polymorphism is G/A, Tan sheep is A, and non-Tan sheep is A or G; the SNP17 is located on the 5972bp of mitochondria, the polymorphism is G/A, Tan sheep is A, and non-Tan sheep is A or G; the SNP18 is located on the 6313bp of mitochondria, the polymorphism is G/A, Tan sheep is A, and non-Tan sheep is A or G; the SNP19 is located on the mitochondria at 13501bp, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP20 is located on the mitochondria at 13551bp, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; the SNP21 is located at 10677bp on mitochondria, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; the SNP22 is located on the mitochondria at 10737bp, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; the SNP23 is located on the 13646bp of mitochondria, the polymorphism is G/A, Tan sheep is G, and non-Tan sheep is A or G; the SNP24 is located at 13863bp on mitochondria, the polymorphism is G/A, Tan sheep is A, and non-Tan sheep is A or G; the SNP25 is located on the 129 th bp of mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP26 is located on the 324bp of mitochondria, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; SNP27 is positioned on the mitochondria at 6674bp, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; the SNP28 is 6839bp on mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP29 is located on the 15194bp of mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP30 is located on the mitochondrion at 15200bp, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP31 is located at 10273bp on mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP32 is located at 10657bp on mitochondria, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; the SNP33 is located on the 10512bp of mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP34 is located on the mitochondria at the 4948bp, the polymorphism is T/G, Tan sheep is T, and non-Tan sheep is G or T; the SNP35 is located on the 16479bp of mitochondria, the polymorphism is C/A, Tan sheep is C, and non-Tan sheep is C or A;
and when the 35 sites of SNP 1-SNP 35 are all Tan sheep genotypes, judging that the sample to be detected contains Tan sheep-derived components, when at least one of the 35 sites is a non-Tan sheep genotype, judging that the sample is a non-Tan sheep, and according to the statistical calculation result of the modeling sample, the judgment accuracy rate is equal to or more than 98% theoretically.
In a third aspect, the present invention provides primers for detecting the SNP marker combination, including 7 pairs of primers, Primer 1-Primer 7, specifically as follows:
the Primer pair Primer1 for detecting SNP1 and SNP2 consists of Primer 1F and Primer 1R, and the Primer sequences are shown as SEQ ID NO 1-2;
the Primer pair Primer 2 for detecting SNP3 and SNP4 consists of Primer 2F and Primer 2R, and the Primer sequences are shown as SEQ ID NO: 3-4;
In a fourth aspect, the invention provides a detection reagent or kit comprising the primers shown in SEQ ID NO. 1-14, or a detection product comprising the primers, probes and the like for detecting the SNP marker combination.
In the fifth aspect, the invention provides the application of the primers shown in SEQ ID NO. 1-14 or the detection reagent or kit containing the primers in the identification, breeding or source component identification of Tan sheep.
In a sixth aspect, the invention provides a method for identifying Tan sheep derived ingredients, comprising the following steps: extracting DNA of a sample to be detected, carrying out real-time fluorescence quantitative PCR by using primers shown in SEQ ID NO. 1-14, detecting genotypes of 14 SNP sites, and judging whether the sample to be detected contains Tan sheep-derived components or not by comparing whether a melting curve peak of an amplification product has a corresponding positive peak type or not or according to a melting curve peak Tm value.
In the above method, it is preferable to add the Primer pairs Primer1 to Primer 4 to the same reaction well or reaction tube, and the Primer pairs Primer 5 to Primer 7 to another reaction well or reaction tube, and to perform multiplex real-time fluorescent quantitative PCR reactions simultaneously or separately. The primers are grouped according to the Tm value of the product. The primers in the group have better specificity and no cross amplification. Different products in the same group correspond to different Tm values, and can be quickly and accurately distinguished by using an RT-PCR melting curve method.
Further, the real-time fluorescent quantitative PCR reaction system is as follows:
reaction system A: 12.5 mu L of Premix of 5 mu M, 0.5-0.6 mu L of each of Primer 1F and Primer 1R, 1.1-1.2 mu L of each of Primer 2F and Primer 2R, 0.9-1.0 mu L of each of Primer 3F and Primer 3R, 0.3-0.4 mu L of each of Primer 4F and Primer 4R, 1 mu L of template DNA of 0.5 ng/mu L, and deionized water to 25 mu L; preferably, reaction system a: premix 12.5. mu.L, 5. mu.M each of Primer 1F and Primer 1R 0.6. mu.L, 5. mu.M each of Primer 2F and Primer 2R 1.2. mu.L, 5. mu.M each of Primer 3F and Primer 3R 1.0. mu.L, 5. mu.M each of Primer 4F and Primer 4R 0.4. mu.L, 0.5 ng/. mu.L of template DNA 1. mu.L, and deionized water to 25. mu.L.
Reaction system B: 12.5 mu L of Premix of 5 mu M, 0.3-0.4 mu L of each of Primer 5F and Primer 5R, 0.3-0.4 mu L of each of Primer 6F and Primer 6R, 0.5-0.6 mu L of each of Primer 7F and Primer 7R, 1 mu L of template DNA of 0.5 ng/mu L, and 25 mu L of deionized water; preferably, reaction system B: premix 12.5. mu.L, 5. mu.M each of Primer 5F and Primer 5R 0.35. mu.L, 5. mu.M each of Primer 6F and Primer 6R 0.4. mu.L, 5. mu.M each of Primer 7F and Primer 7R 0.5. mu.L, 0.5 ng/. mu.L of template DNA 1. mu.L, and deionized water to make up to 25. mu.L.
The real-time fluorescent quantitative PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing at 62 ℃ and elongation for 45s for 30 cycles.
The melting curve was prepared under the following conditions: the temperature is raised to 90 ℃ at the speed of 0.02 ℃/s for 1min at 95 ℃ and 1min at 67 ℃, and the fluorescence intensity is continuously detected.
According to the method, whether the sample to be detected contains Tan sheep-derived ingredients or not is judged according to the number of melting curve peaks and whether the melting curve peaks appear at corresponding positions;
the Tm value of an amplification product corresponding to the Primer pair Primer1 is 71.6 +/-0.6 ℃, the Tm value of an amplification product corresponding to the Primer pair Primer 2 is 74.5 +/-0.6 ℃, the Tm value of an amplification product corresponding to the Primer pair Primer 3 is 79.8 +/-0.8 ℃, the Tm value of an amplification product corresponding to the Primer pair Primer 4 is 81.5 +/-0.7 ℃, the Tm value of an amplification product corresponding to the Primer pair Primer 5 is 76.8 +/-0.8 ℃, the Tm value of an amplification product corresponding to the Primer pair Primer 6 is 79.6 +/-0.5 ℃, and the Tm value of an amplification product corresponding to the Primer pair Primer 7 is 81.1 +/-0.6 ℃.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention provides a site combination consisting of 14 mitochondrial Single Nucleotide Polymorphism (SNP) sites, a multiple real-time fluorescent chain polymerase reaction (RT-PCR) melting curve identification method and a kit for identifying Tan sheep. The invention establishes an RT-PCR system based on SNP sites developed on mitochondria, mitochondrial DNA is maternal inheritance, the evolution rate is high, non-coding regions are few, the mutation rate is moderate, and the like, has certain polymorphism and traceability, and can be used as a marker gene. The method provides a guarantee foundation for identifying the truth of Tan sheep, and has important significance for improving the whole brand value of Tan sheep industry and protecting precious sheep species and breeding and processing enterprises thereof. According to the method, the specific variation sites in the Tan sheep mitochondrial gene are screened by sequencing the mitochondrial gene, whether the melting curve peaks of the RT-PCR products of the two systems have corresponding peak types or not is compared, whether the detection sample is a Tan sheep sample or not is judged, and technical support is provided for authenticity identification and genetic breeding of Tan sheep. The method is quick and simple to operate, intuitive and reliable in result and low in cost, and has important significance for preventing adulteration and counterfeiting, improving the overall brand value of the Tan sheep industry and promoting healthy and rapid development of the industry.
Drawings
FIG. 1 is a total DNA electrophoresis chart of Tan sheep and non-Tan sheep species (10 randomly selected) in the preferred embodiment of the present invention; wherein, 1-10: randomly selecting 10 Tan sheep and non-Tan sheep genome DNAs, M: and (5) DNA Marker.
FIG. 2 is a diagram of the peak of the melting curve of positive sample of Tan sheep in the quadruple RT-PCR system in the preferred embodiment of the present invention.
FIG. 3 is a peak diagram of the melting curve of Tan sheep positive sample in the triple RT-PCR system in the preferred embodiment of the present invention.
FIGS. 4-5 are graphs of non-Tan sheep species-specific melting curve verification peaks in RT-PCR system according to the preferred embodiment of the present invention.
FIGS. 6-7 are graphs of melting curve peaks detected from commercially available samples in RT-PCR system according to a preferred embodiment of the present invention.
Detailed Description
The invention provides an application of a reagent for detecting the genotype of the following 14 SNP site combinations in the mitochondria of a Tan sheep to be detected in identification or auxiliary identification of the sheep to be detected as the Tan sheep or a non-Tan sheep; whether the detected sample is a Tan sheep sample can be judged by comparing whether the melting curve peaks of the RT-PCR products of the two systems have corresponding peak types.
The invention adopts the following technical scheme:
the physical position of each SNP locus in the 14 SNP locus combinations is determined based on the alignment of Tan sheep whole mitochondria standard sequence (GenBank: KF938336.1, 16618bp altogether; PUBMED: 26085518). The 14 SNP loci are respectively: the SNP1 is located on the 932 th bp of mitochondria, the polymorphism is A/G, Tan sheep is A, and non-Tan sheep is A or G; SNP2 is located at 955bp on mitochondria, polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP3 is located on the 7301bp of mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP4 is located on the 7350bp of mitochondria, the polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP5 is located on the upper 12645bp of mitochondria, the polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP6 is positioned on the 12813bp of mitochondria, the polymorphism is G/A, Tan sheep is G, and non-Tan sheep is A or G; the SNP7 is located at 14873bp on mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP8 is located at 14951bp on mitochondria, the polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T. The SNP9 is located at 10954bp on mitochondria, the polymorphism is C/T, Tan sheep is C, non-Tan sheep is C or T, the SNP10 is located at 11031bp on mitochondria, the polymorphism is C/T, Tan sheep is C, non-Tan sheep is C or T; the SNP11 is located on the 1371bp of mitochondria, the polymorphism is A/G, Tan sheep is A, and non-Tan sheep is A or G; the SNP12 is located at the 1512bp on mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP13 is located on the 8712bp of mitochondria, the polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP14 is located at the 8866bp on mitochondria, the polymorphism is G/A, Tan sheep is G, and non-Tan sheep is A or G
In the application, the reagent for detecting the combined genotypes of the following 14 SNP loci in the genome of the sheep to be detected comprises the following biological materials:
1. the primers for amplifying the 14 SNP sites are shown in Table 1(SEQ ID NOS: 1-14).
TABLE 1
2. Primer combinations Primer1 to Primer 4 are placed in the reaction hole A, Primer combinations Primer 5 to Primer 7 are placed in the reaction hole B, the total volume of each reaction system is 25 mu l (table 2), and amplification is carried out according to the RT-PCR reaction conditions in the table 3.
TABLE 2 RT-PCR reaction System
TABLE 3 RT-PCR reaction conditions
And further comparing whether the melting curve peaks of the RT-PCR products of the two reaction holes have corresponding peak types or not, wherein Tm values of amplification products of the specific primers in the reaction holes A or B have significant difference, and judging whether the detection sample is a Tan sheep sample or not by using a melting curve method.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The main apparatus comprises: real-time fluorescent quantitative PCR instrument (Roche480 Switzerland), high-speed desktop centrifuge (Eppendorf5417R, Germany), micropipette (2.5. mu.L, 10. mu.L, 100. mu.L, 1000. mu.L), homogenizer (Omni Prep, USA), luciferase reader (Bio tek Synergy H4, USA), and the like.
The main reagents are as follows: DNA extraction kits for blood and animal tissue were purchased from Qiagen; premix available from Roche; the primers were synthesized by Enwei fundi (Shanghai) trade Co., Ltd.
Example 1 ability of SNP site combination to discriminate Tan sheep variety
Genomic DNAs of 100 Tan sheep and 44 non-Tan sheep are extracted by a CTAB method, and quality inspection is carried out by agarose gel electrophoresis, wherein the electrophoresis band of the genomic DNA is usually not less than 20kb and has no dispersion (figure 1). And amplifying and sequencing the DNA qualified by quality inspection through PCR to obtain 144 sheep mitochondrial gene sequences. And simultaneously, downloading 19 non-Tan sheep mitochondrial genes through an NCBI website (https:// www.ncbi.nlm.nih.gov /) to obtain 163 mitochondrial DNA sequence information of Tan sheep and non-Tan sheep samples in total.
And (3) processing the collected 163 mitochondrial gene information through a Python program and Mega software to construct a Tan sheep identification method. Selecting sites of SNP sites which are conserved in species of Tan sheep and have high polymorphism of other varieties, selecting 35 sites from 399 sites as characteristic markers for identifying whether the sites are Tan sheep samples, wherein the physical positions of the SNP sites are determined based on comparison of Tan sheep whole mitochondrial standard sequences (GenBank: KF 938336.1). When the 35 sites are all Tan sheep genotypes, the sample to be detected is judged to contain Tan sheep derived components, when at least one of the 35 sites is a non-Tan sheep genotype, the sample to be detected is judged to be a non-Tan sheep, and according to the statistical calculation result of the modeling sample, the accuracy rate of the method is not less than 98% theoretically (Table 4).
TABLE 4 Tan sheep identification method theoretical accuracy
EXAMPLE 2 establishment of triple and quadruple RT-PCR reaction systems and investigation of specificity
1. SNP site primer design
PCR amplification primers for the SNP site to be detected were designed using Oligo 7.0 software (Table 1) and handed over to Yinxie funding Co.
2. Establishment of RT-PCR reaction System
Combining primers according to the reaction system A: premix 12.5. mu.L, 5. mu.M each of Primer 1F and Primer 1R 0.3-1.5. mu.L, 5. mu.M each of Primer 2F and Primer 2R 0.3-1.5. mu.L, 5. mu.M each of Primer 3F and Primer 3R 0.3-1.5. mu.L, 5. mu.M each of Primer 4F and Primer 4R 0.3-1.5. mu.L, 0.5 ng/. mu.L of template DNA 1. mu.L, deionized water to 25. mu.L;
reaction system B: premix 12.5. mu.L, 5. mu.M Primer 5F and Primer 5R each 0.3-1.5. mu.L, 5. mu.M Primer 6F and Primer 6R each 0.3-1.5. mu.L, 5. mu.M Primer 7F and Primer 7R each 0.3-1.5. mu.L, 0.5 ng/. mu.L template DNA 1. mu.L, and deionized water to make up to 25. mu.L.
Under the condition of ensuring unimodal specificity, different primer combinations are constructed for RT-PCR so as to improve the discrimination of melting curve peak shapes in a multiple system.
Finally, the reaction system A and B is obtained through optimization as follows:
combining primers according to the reaction system A: premix 12.5. mu.L of Premix 5. mu.M of each of Primer 1F and Primer 1R 0.6. mu.L, 5. mu.M of each of Primer 2F and Primer 2R 1.2. mu.L, 5. mu.M of each of Primer 3F and Primer 3R 1.0. mu.L, 5. mu.M of each of Primer 4F and Primer 4R 0.4. mu.L, 0.5 ng/. mu.L of template DNA 1. mu.L, and deionized water to 25. mu.L;
reaction system B: premix 12.5. mu.L, 5. mu.M each of Primer 5F and Primer 5R 0.35. mu.L, 5. mu.M each of Primer 6F and Primer 6R 0.4. mu.L, 5. mu.M each of Primer 7F and Primer 7R 0.5. mu.L, 0.5 ng/. mu.L of template DNA 1. mu.L, and deionized water to make up to 25. mu.L.
3. Conditions for RT-PCR reaction
A. And B, synchronously amplifying the two reaction holes on an RT-PCR instrument according to the reaction conditions in the table 3.
4. Melting curve analysis RT-PCR amplification product Tm value
Reading fluorescence signals after each cycle of the amplification link, and analyzing the specificity of triple and quadruple RT-PCR of A and B reaction wells by a melting curve method, wherein the ratio of A reaction well: 4 corresponding peak shapes (figure 2 and figure 3) should appear on the Tan sheep sample, and any one or more than 4 amplification peaks in the Tan sheep sample should be deleted; b, reaction hole: the Tan sheep sample should have 3 corresponding peak shapes, and the non-Tan sheep sample should lack any one or more than 3 amplification peaks in the peak shapes. The detection results are shown in fig. 4 and 5, and the detected sheep species all accord with the corresponding peak shapes.
Example 3 sample testing and method accuracy review
1. Collecting sample, preparing template
60 parts of DNA of samples of Tan sheep and commercial non-Tan sheep are extracted by a CTAB method. DNA was extracted from Tan sheep and non-Tan sheep, respectively, and the concentration and purity of the DNA were calculated according to the DNA extraction method described in reference example 1. The DNA samples were diluted to 0.5 ng/. mu.L each and used as detection templates.
2. Triple and quadruple RT-PCR reaction verification
Referring to the RT-PCR reaction process in example 2, 120 samples of Tan sheep and non-Tan sheep DNA templates were subjected to triple and quadruple RT-PCR dissolution curve analysis in two reaction wells simultaneously or respectively, and Tm values were calculated and the number of melting curve peaks was counted.
3. Sample validation
Determination standard of Tan sheep: by performing triple and quadruple RT-PCR dissolution curve analysis on 60 Tan sheep samples and 60 non-Tan sheep samples, 7 melting curve peaks appear, the Tm value accords with the value range, and the Tan sheep can be judged, and the non-Tan sheep can be judged uniformly when the Tm value is less than 7 melting curve peaks (fig. 6 and 7). Through the judgment of the samples, all 60 Tan sheep samples are judged as Tan sheep, and of the 60 non-Tan sheep samples, 42 samples are judged as non-Tan sheep, and 18 samples are judged as Tan sheep by mistake, so that the final accuracy is 85% (table 5), and the method is proved to have higher accuracy and practicability.
TABLE 5 identification of SNP site results in Tan and non-Tan sheep
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> China meat food comprehensive research center
<120> identification method of Tan sheep derived components
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Claims (10)
1. The core SNP marker combination for identifying Tan sheep is characterized by comprising 14 SNP loci including SNP 1-SNP 14, wherein the physical position of each SNP locus is determined by referring to Tan sheep mitochondrial genome sequence GenBank KF938336.1 as follows: the SNP1 is located on the 932 th bp of mitochondria, the polymorphism is A/G, Tan sheep is A, and non-Tan sheep is A or G; SNP2 is located at 955bp on mitochondria, polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP3 is located on the 7301bp of mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP4 is located on the 7350bp of mitochondria, the polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP5 is located on the upper 12645bp of mitochondria, the polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP6 is positioned on the 12813bp of mitochondria, the polymorphism is G/A, Tan sheep is G, and non-Tan sheep is A or G; the SNP7 is located at 14873bp on mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP8 is located at 14951bp on mitochondria, the polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP9 is located at 10954bp on mitochondria, the polymorphism is C/T, Tan sheep is C, non-Tan sheep is C or T, the SNP10 is located at 11031bp on mitochondria, the polymorphism is C/T, Tan sheep is C, non-Tan sheep is C or T; the SNP11 is located on the 1371bp of mitochondria, the polymorphism is A/G, Tan sheep is A, and non-Tan sheep is A or G; the SNP12 is located at the 1512bp on mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP13 is located on the 8712bp of mitochondria, the polymorphism is T/C, Tan sheep is T, and non-Tan sheep is C or T; the SNP14 is located at the 8866bp on mitochondria, the polymorphism is G/A, Tan sheep is G, and non-Tan sheep is A or G.
2. The SNP marker combination for identifying Tan sheep is characterized by comprising 14 core SNP markers and 21 extension SNP markers; wherein, the 14 core SNP markers comprise 14 SNP sites of SNP 1-SNP 14, and the 14 SNP sites are as defined in claim 1; the 21 extended SNP markers comprise 21 SNP sites of SNP 15-SNP 35;
for SNPs 15-35, the physical position of each SNP site is determined by referring to Tan sheep mitochondrial genome sequence GenBank KF938336.1 as follows: the SNP15 is located on the 3037bp of mitochondria, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; the SNP16 is positioned on the mitochondria at 3299bp, the polymorphism is G/A, Tan sheep is A, and non-Tan sheep is A or G; the SNP17 is located on the 5972bp of mitochondria, the polymorphism is G/A, Tan sheep is A, and non-Tan sheep is A or G; the SNP18 is located on the 6313bp of mitochondria, the polymorphism is G/A, Tan sheep is A, and non-Tan sheep is A or G; the SNP19 is located on the mitochondria at 13501bp, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP20 is located on the mitochondria at 13551bp, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; the SNP21 is located at 10677bp on mitochondria, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; the SNP22 is located on the mitochondria at 10737bp, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; the SNP23 is located on the 13646bp of mitochondria, the polymorphism is G/A, Tan sheep is G, and non-Tan sheep is A or G; the SNP24 is located at 13863bp on mitochondria, the polymorphism is G/A, Tan sheep is A, and non-Tan sheep is A or G; the SNP25 is located on the 129 th bp of mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP26 is located on the 324bp of mitochondria, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; SNP27 is positioned on the mitochondria at 6674bp, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; the SNP28 is 6839bp on mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP29 is located on the 15194bp of mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP30 is located on the mitochondrion at 15200bp, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP31 is located at 10273bp on mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP32 is located at 10657bp on mitochondria, the polymorphism is C/T, Tan sheep is T, and non-Tan sheep is C or T; the SNP33 is located on the 10512bp of mitochondria, the polymorphism is C/T, Tan sheep is C, and non-Tan sheep is C or T; the SNP34 is located on the mitochondria at the 4948bp, the polymorphism is T/G, Tan sheep is T, and non-Tan sheep is G or T; the SNP35 is located on the 16479bp of mitochondria, the polymorphism is C/A, Tan sheep is C, and non-Tan sheep is C or A.
3. The primers for detecting the SNP marker combination according to claim 1, wherein the Primer comprises 7 Primer pairs of Primer 1-Primer 7, and the Primer pairs specifically comprise:
the Primer pair Primer1 for detecting SNP1 and SNP2 consists of Primer 1F and Primer 1R, and the Primer sequences are shown as SEQ ID NO 1-2;
the Primer pair Primer 2 for detecting SNP3 and SNP4 consists of Primer 2F and Primer 2R, and the Primer sequences are shown as SEQ ID NO: 3-4;
primer 3 for detecting SNP5 and SNP6, which consists of Primer 3F and Primer 3R, and the Primer sequences are shown as SEQ ID NO: 5-6;
primer 4 for detecting SNP7 and SNP8, which consists of Primer 4F and Primer 4R, and the Primer sequences are shown as SEQ ID NO: 7-8;
primer 5 for detecting SNP9 and SNP10, which consists of Primer 5F and Primer 5R, and the Primer sequences are shown as SEQ ID NO. 9-10;
primer 6 for detecting SNP11 and SNP12, which consists of Primer 6F and Primer 6R, and the Primer sequences are shown as SEQ ID NO: 11-12;
primer 7 for detecting SNP13 and SNP14 consists of Primer 7F and Primer 7R, and the Primer sequences are shown as SEQ ID NO 13-14.
4. A detection reagent or kit comprising the primer according to claim 3, or a detection product comprising the primer and the probe for detecting the SNP marker combination according to claim 1 or 2.
5. The primer of claim 3 or the detection reagent or the kit of claim 4, wherein the primer is used for identification, breeding or identification of Tan sheep-derived components.
6. The identification method of Tan sheep derived components is characterized by comprising the following steps: extracting DNA of a sample to be detected, carrying out real-time fluorescence quantitative PCR by using the primer of claim 2, detecting genotypes of 14 SNP sites, and judging whether the sample to be detected contains Tan sheep-derived components or not by comparing whether a melting curve peak of an amplification product has a corresponding positive peak type or not or according to a melting curve peak Tm value.
7. The method according to claim 6, characterized in that Primer pairs Primer1 to Primer 4 are added to the same reaction well or reaction tube, Primer pairs Primer 5 to Primer 7 are added to another reaction well or reaction tube, and multiple real-time fluorescent quantitative PCR reactions are performed simultaneously or separately.
8. The method of claim 7, wherein the real-time fluorescent quantitative PCR reaction system comprises:
reaction system A: 12.5 mu L of Premix of 5 mu M, 0.5-0.6 mu L of each of Primer 1F and Primer 1R, 1.1-1.2 mu L of each of Primer 2F and Primer 2R, 0.9-1.0 mu L of each of Primer 3F and Primer 3R, 0.3-0.4 mu L of each of Primer 4F and Primer 4R, 1 mu L of template DNA of 0.5 ng/mu L, and deionized water to 25 mu L;
reaction system B: 12.5. mu.L of Premix 5. mu.M, 0.3 to 0.4. mu.L each of Primer 5F and Primer 5R, 0.3 to 0.4. mu.L each of Primer 6F and Primer 6R, 0.5 to 0.6. mu.L each of Primer 7F and Primer 7R, 1. mu.L of template DNA at 0.5 ng/. mu.L, and 25. mu.L of deionized water.
9. The method of claim 7, wherein the real-time fluorescent quantitative PCR reaction conditions are: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing at 62 ℃ and extension for 45s for 30 cycles;
the melting curve was prepared under the following conditions: the temperature is raised to 90 ℃ at the speed of 0.02 ℃/s for 1min at 95 ℃ and 1min at 67 ℃, and the fluorescence intensity is continuously detected.
10. The method according to any one of claims 6 to 9, wherein whether the sample to be tested contains Tan sheep derived components is determined according to the number of melting curve peaks and whether the melting curve peaks appear at corresponding positions;
the Tm value of an amplification product corresponding to the Primer pair Primer1 is 71.6 +/-0.6 ℃, the Tm value of an amplification product corresponding to the Primer pair Primer 2 is 74.5 +/-0.6 ℃, the Tm value of an amplification product corresponding to the Primer pair Primer 3 is 79.8 +/-0.8 ℃, the Tm value of an amplification product corresponding to the Primer pair Primer 4 is 81.5 +/-0.7 ℃, the Tm value of an amplification product corresponding to the Primer pair Primer 5 is 76.8 +/-0.8 ℃, the Tm value of an amplification product corresponding to the Primer pair Primer 6 is 79.6 +/-0.5 ℃, and the Tm value of an amplification product corresponding to the Primer pair Primer 7 is 81.1 +/-0.6 ℃.
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| WO2008056325A2 (en) * | 2006-11-06 | 2008-05-15 | Universidade Do Porto | Process for animal species identification in samples with genetic material based on mitochondrial dna size variation |
| CN109182544A (en) * | 2018-10-18 | 2019-01-11 | 北京康普森生物技术有限公司 | A kind of 8 kinds of SNP sites and its application for identifying sheep known for its fine thick wool and non-sheep known for its fine thick wool |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008056325A2 (en) * | 2006-11-06 | 2008-05-15 | Universidade Do Porto | Process for animal species identification in samples with genetic material based on mitochondrial dna size variation |
| CN109182544A (en) * | 2018-10-18 | 2019-01-11 | 北京康普森生物技术有限公司 | A kind of 8 kinds of SNP sites and its application for identifying sheep known for its fine thick wool and non-sheep known for its fine thick wool |
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| 马丽娜等: "宁夏滩羊mtDNA Cyt b基因遗传多样性分析", 《黑龙江畜牧兽医》 * |
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