CN113832236A - Primer group and kit for identifying sika deer, red deer and hybrid deer and application - Google Patents
Primer group and kit for identifying sika deer, red deer and hybrid deer and application Download PDFInfo
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Abstract
The invention discloses a primer group, a kit and application for identifying sika deer, red deer and hybrid deer, and belongs to the technical field of biology. The primer group comprises a primer group shown as SEQ ID NO: 1 to SEQ ID NO: 400, or a pharmaceutically acceptable salt thereof. Use of the MNP marker site, the use comprising: 200 MNP marker sites as shown in Table 1 of the specification are used for genetic difference analysis of sika deer, red deer and hybrid deer and blood lineage analysis of sika deer, red deer and hybrid deer. The primer group provided by the embodiment of the invention has good discrimination, reproducibility and accuracy, and the kit using the primer group can simultaneously have the advantages and can effectively ensure the detection accuracy.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer group, a kit and application for identifying sika deer, red deer and hybrid deer.
Background
The pilose antler is a traditional and more valuable Chinese medicinal material in China, has obvious health care curative effects on enhancing the body functions and eliminating fatigue, and is widely applied to clinical medicine. The definition in pharmacopoeia of the people's republic of china (2015 edition): the cornu Cervi Pantotrichum primordium is young horn of non-ossified dense cornu Cervi Pantotrichum of Cervidae Cervus Nippon Temminck or Cervus Elaphus Linnaeus. At present, researches show that the contents of amino acid, nucleoside components, phospholipid components, cholesterol and polyamine in the velvet antlers of the spotted deer and the velvet antlers of the red deer are different, wherein the content of the nucleoside components in the velvet antlers of the spotted deer is obviously higher than that of the velvet antlers of the red deer, which may be the reason for different medicinal values of the two velvet antlers. In addition, the quality of the spotted deer antler harvested by the northeast sika deer is generally considered to be the best, the quality of the east mashroom antler harvested by the northeast maka deer is better, and the quality of the west mashroom antler produced in northwest of China is inferior. Therefore, the spotted deer antler is more valuable in the market, and the price of the spotted deer antler is about 3 times higher than that of the red deer antler. Because the spotted deer antler is expensive, the phenomenon that spotted deer antler is replaced by the spotted deer antler or the antler of a hybrid deer bred by spotted deer is common in China, the standardization of the traditional Chinese medicine market and the development of the industry are seriously influenced, and the legal rights and interests of consumers are infringed. Therefore, the establishment of an accurate and efficient antler identification method has important significance.
At present, the method for identifying the truth of pilose antler and mixed counterfeit products thereof mostly adopts PCR (Polymerase Chain Reaction) technology to detect COI gene of mitochondria or SRY gene on Y chromosome, electrophoresis is carried out after the COI gene of mitochondria is amplified to obtain amplified products, and the sources of pilose antler are distinguished by identifying the size and shape of the amplified products. Since mitochondria are from female parent, a series of identification methods based on COI gene can only be used as the basis for identifying female parent, and for hybrid deer, female parent can be sika deer, and father can be red deer or deer of other varieties, which makes the method unable to accurately identify the antler of hybrid deer whose father is non-sika deer.
Disclosure of Invention
In order to solve the problems in the prior art, the embodiment of the invention provides a primer group, a kit and application for identifying sika deer, red deer and hybrid deer. The technical scheme is as follows:
in one aspect, the invention provides a primer set for identifying sika deer, red deer and hybrid deer, wherein the primer set comprises the nucleotide sequence shown in SEQ ID NO: 1 to SEQ ID NO: 400, or a pharmaceutically acceptable salt thereof.
In still another aspect, the present embodiments provide a kit for identifying sika deer, red deer and hybrid deer, the kit comprising the primer set of claim 1.
In yet another aspect, embodiments of the present invention provide an application of MNP marker loci, including: 200 MNP marker sites as shown in Table 1 of the specification were used for genetic difference analysis of sika deer, red deer and hybrid deer and blood lineage analysis of sika deer, red deer and hybrid deer.
The technical scheme provided by the embodiment of the invention has the following beneficial effects: the primer group provided by the embodiment of the invention is used for identifying sika deer, red deer and hybrid deer, utilizes multiple PCR amplification, fuses a second-generation sequencing platform for sequencing of amplification products, realizes the advantages of multiple targets, high flux, high efficiency and high accuracy for detecting the deer antlers, has high individual discrimination and high reproducibility and accuracy, and can detect multiple targets by utilizing multiple PCR at one time, thereby effectively avoiding the problems of high false negative and low sensitivity caused by the amplification failure of a single target. The kit using the primer group can simultaneously have the advantages, and can effectively ensure the reproducibility and accuracy of detection. The MNP marker loci are used for genetic difference analysis of the sika deer, the red deer and the hybrid deer and blood lineage analysis of the sika deer, the red deer and the hybrid deer, alleles of the MNP marker loci are rich and have high polymorphism, 2n allelic forms exist on a single MNP marker locus and are higher than SSR and SNP, the species distinguishing capability of the MNP marker loci is high, species can be distinguished on the species level due to the fact that a plurality of MNP marker loci can be identified simultaneously, different individuals in the species can be identified, the sika deer, the red deer and the hybrid deer can be identified effectively, and technical support is provided for standardized management of the velvet industry and genetic research of the sika deer and the red deer.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a clustering tree diagram according to an embodiment of the present invention;
FIG. 2 is a clustering distribution chart according to a second embodiment of the present invention;
fig. 3 is a diagram of analyzing the blood lineage of sika deer according to the third embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below.
Example one
The embodiment of the invention provides a primer group for identifying sika deer, red deer and hybrid deer, which comprises the following components in percentage by weight: the primer group comprises an upstream primer and a downstream primer, and specifically comprises a primer group shown as SEQ ID NO: 1 to SEQ ID NO: 400, or a pharmaceutically acceptable salt thereof.
The genome sequencing data (receiving number: PRJNA355630) of 100 sika deer and 68 sika deer individuals obtained from SRA database of NCBI are combined by taking publicly released sika genome sequence GCA _002197005.1 as a reference genome, adopting Samtools (Version 1.2) and BCFtools (Version 1.2) to identify SNP sites on the genome, carrying out comparative analysis with NT database of NCBI, and carrying out screening of MNP markers according to the following principle:
(1) the marker sequence is shared by sika deer and red deer, but does not appear in other species;
(2) a plurality of discrete SNP differences in sequence;
(3) the length of the marker sequence is between 200 and 300 bp.
And further screening the MNP marker loci from the sequencing data of the known sika deer and red deer by comparison, and finally screening 200 MNP marker loci.
In use, SEQ ID NO: 1 to SEQ ID NO: the primers shown in 400 were mixed in equal amounts. Specifically, SEQ ID NO: 1 to SEQ ID NO: 5. mu.L of each of the primers shown in 400 was mixed and used.
In another aspect, the embodiment of the invention provides a kit for identifying sika deer, red deer and hybrid deer, which comprises the primer set.
The performance of the primer set was evaluated as follows:
the primer group provided by the embodiment of the invention is adopted to respectively test the MNP marker sites of 4 spotted deer DNA samples and 4 red deer antler DNA samples provided by the Chinese inspection and quarantine scientific research institute, and the 4 spotted deer antler DNA samples and the 4 red deer antler DNA sources provided by the embodiment of the invention are both given by the Chinese inspection and quarantine scientific research institute. Specifically, the primer set provided by the embodiment of the invention is adopted to perform multiplex PCR amplification on 8 DNA samples, the number of amplification cycles is designed to be 20, amplification products are obtained, a high-throughput sequencing library is constructed by adopting the amplification products, a MiSeq platform of illumina is adopted to perform second-generation sequencing on the high-throughput sequencing library, sequencing fragment sets are obtained, and analysis and statistics results are respectively obtained according to 200 MNP (MNP) labeled sites shown in Table 1.
Table 1 shows 200 MNP marker loci and corresponding primer sequence table
In order to verify the accuracy of the result, each DNA sample is divided into two parts before detection, 16 libraries are respectively constructed at two different times (the time interval between two times is 7 days) and subjected to second-generation sequencing to obtain 16 sequencing fragment groups, and the typing results of the 16 sequencing fragment groups are respectively analyzed and counted according to 200 MNP marker sites shown in table 1, wherein the specific results are shown in table 2.
Table 2 shows the evaluation information table of 200 MNP marker loci
As can be seen from the combination of the table 2, at least 194 MNP marker sites are detected from the sika deer DNA sample and the red deer DNA sample, the site detection rate exceeds 97%, the average detection rate of the obtained 16 high-throughput sequencing libraries reaches 98.13%, and the primer group can efficiently detect the screened MNP marker sites, has strong primer specificity and high primer amplification efficiency; comparing the typing results of the MNP marker sites detected in the high-throughput sequencing library repeatedly constructed by each sample, the repeated typing results of 3140 MNP marker sites are completely the same, and therefore, the repeatability is 100%, and the accuracy is 100%. Comparing the typing results of the MNP marker loci among the samples, according to the principle that the MNP marker loci are different when at least 1 SNP difference exists among the samples, 94.33% of the MNP marker loci are different between the DNA sample of the deer antler of spotted deer and the DNA sample of the deer antler of spotted deer, 10.25% of the MNP marker loci are different among individual spotted deer, and the clustering analysis result is shown in figure 1. The result shows that the 200 MNP marker loci provided by the embodiment of the invention have high detection rate, high typing accuracy and high variety discrimination, and can obviously distinguish the deer antlers from sika deer and red deer. Meanwhile, the MNP marker loci have rich alleles and high polymorphism, 2n allelic types are arranged on a single MNP marker locus, the allelic types are higher than those of SSR and SNP, the species distinguishing capability of the MNP marker loci is high, and due to the fact that a plurality of MNP marker loci can be identified at the same time, the species can be distinguished on the species level, and different individuals in the species can be identified. Moreover, the efficiency of MNP marker locus detection is high, and tens of thousands of MNP marker loci of hundreds of samples can be typed at one time by adopting the combination of ultra-multiplex PCR and the second-generation high-throughput sequencing technology to detect the MNP marker loci. In addition, the primer group provided by the embodiment of the invention has high site amplification specificity and high detectable rate, multiple targets can be detected by utilizing multiple PCR (polymerase chain reaction) at one time, so that the problems of high false negative and low sensitivity caused by the failure of amplification of a single target can be effectively solved, meanwhile, the primer group has high detection accuracy, the primer group is matched with a second-generation high-throughput sequencer to sequence an amplification product for hundreds of times, and the output result is a base sequence, so that parallel experiments are not needed, data can be randomly compared, and the data sharing is strong.
Example two
The embodiment of the invention provides an application of MNP marker loci, which comprises the following steps: the application comprises the following steps: the nucleotide sequence shown as SEQ ID NO: 401 to SEQ ID NO: the MNP marker locus shown in 600 is used for genetic difference analysis of sika deer, red deer and hybrid deer and blood lineage analysis of sika deer, red deer and hybrid deer.
The 200 MNP marker loci and the kit provided by the embodiment of the invention are used for carrying out MNP marker locus difference analysis on 29 sika deer, 32 red deer and 7 hybrid deer F1 generations submitted by the scientific research institute of Chinese inspection and quarantine, 68 deer individual DNA in total, and all samples are found to be divided into three categories of sika deer, sika deer and hybrid deer through clustering analysis, and the results are shown in figure 2. Wherein 14 stags, 2 stags and hybrid deer are gathered into one type, the sample source is supposed to be caused by wrong sample source during sample collection, the result is consistent with the main component PCoA analysis result by using MNP marker difference among samples, and the MNP marker locus can be used for genetic difference analysis of the stags, the stags and the hybrid deer. The sika deer, the red deer and the hybrid deer are respectively self-assembled into one type, and can obviously distinguish individuals among groups and individuals in the groups.
Example 3 analysis of the blood systems of Cervus Nippon Temminck, Cervus Elaphus L and hybrid deer
Except for the sample sequencing data with inconsistent sample names and classifications in example 2, the MNP marker typing data of the remaining 26 sika deer and 25 red deer is analyzed, the specific MNP alleles only existing in the sika deer are screened, 78 MNP marker loci are obtained in total, the number (T) of the sika deer specific alleles on the 78 loci of the samples is analyzed, the ratio of the sika deer specific alleles in each sample is calculated, and a sika deer blood lineage calculation method is established, namely the sika deer blood lineage ratio (M) is the number (T) of the sika deer specific alleles/the total number (N) of the 78 MNP marker alleles, wherein the higher M value is the closer to the sika deer blood margin relation. The method is used for analyzing the sika deer blood lineage of 65 deer individuals in the embodiment 2, the analysis result is shown in fig. 3, as can be seen from fig. 3, the average value of the sika deer individual sample M is 1 (namely all sika deer blood lineage), the average value of the red deer M is 0 (namely all sika deer blood lineage are not contained), and the average value of the hybrid deer F1 is 0.46 (namely about half of sika deer blood lineage is contained), and the result shows that the sika deer blood lineage determined by the sika deer blood lineage calculation method established by the embodiment of the invention is consistent with the actual situation and has high accuracy. In addition, the genetic difference clustering in example 2 shows that the M mean value of 13 elk samples of the hybrid deer is 0.71, which indicates that the elk samples indeed contain sika deer blood margin, belong to the type of the hybrid deer and are consistent with the clustering result.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (3)
1. A primer group for identifying sika deer, red deer and hybrid deer is characterized by comprising the following components in sequence table as shown in SEQ ID NO: 1 to SEQ ID NO: 400, or a pharmaceutically acceptable salt thereof.
2. A kit for identifying sika deer, red deer and hybrid deer, comprising the primer set of claim 1.
3. Use of a MNP marker site, comprising: 200 MNP marker sites as shown in Table 1 of the specification are used for genetic difference analysis of sika deer, red deer and hybrid deer and blood lineage analysis of sika deer, red deer and hybrid deer.
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Cited By (2)
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| CN113122644A (en) * | 2021-05-31 | 2021-07-16 | 中国农业科学院特产研究所 | SNP (Single nucleotide polymorphism) locus for detecting blood source content of red deer, screening method, corresponding SNP chip and application |
| CN114672575A (en) * | 2022-04-19 | 2022-06-28 | 中国检验检疫科学研究院 | Kit for rapidly detecting hybrid antler based on RAA amplification |
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| CN114672575A (en) * | 2022-04-19 | 2022-06-28 | 中国检验检疫科学研究院 | Kit for rapidly detecting hybrid antler based on RAA amplification |
| CN114672575B (en) * | 2022-04-19 | 2024-04-12 | 中国检验检疫科学研究院 | Kit for rapidly detecting hybridized pilose antler based on RAA amplification |
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