MNP marker site for poultry identification, primer composition, reagent, kit and application

Abstract

The application relates to the field of biotechnology, in particular to an MNP marker site for identifying poultry, a primer composition, a reagent, a kit and application; the MNP marker site is a genomic region with species specificity and multiple nucleotide polymorphisms in the species, which is screened on the genome of each kind of poultry; the MNP marker site comprises 15 marker sites of MNP-1 to MNP-15; and a multiplex primer composition is designed according to the found MNP marker site, the designed multiplex primer composition can be used for multiplex PCR amplification, and a second-generation sequencing platform is fused to perform sequencing of the amplification products, so that one-time identification and differentiation of five kinds of poultry and meat products with high throughput, high efficiency and high accuracy are met, and technical support is provided for solving the meat adulteration problem.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to an MNP marker site, primer composition, reagent, kit, and application for the identification of poultry. Background Technology

[0002] Chicken, duck, goose, pigeon, and quail are five common poultry species, and their meat products account for a large proportion of food consumption. In recent years, the problem of adulteration in meat products has become increasingly serious, making the identification and traceability analysis of adulterated meat products a research hotspot. Traditional methods of meat identification based on sensory and morphological methods are no longer sufficient to meet the needs of identifying adulterated meat. Protein-based detection methods, such as immunoassay and chromatographic analysis, are not suitable for processed meat products. DNA-based detection methods can detect adulterated meat products, with fluorescent PCR being a commonly used method. However, fluorescent PCR can only detect one marker for one type of poultry at a time, requiring multiple tests to distinguish between five types of poultry. Furthermore, variations in the marker primer region can lead to detection failure, resulting in limitations in detection efficiency and accuracy.

[0003] Therefore, how to provide multiple novel molecular markers with high polymorphism to achieve efficient, accurate and sensitive detection of multiple markers in five poultry species in a single test, thereby enabling accurate identification and differentiation of five poultry meat products, is a technical problem that urgently needs to be solved. Summary of the Invention

[0004] This application provides an MNP marker site, primer composition, reagent, kit, and application for poultry identification, to solve the technical problem in the prior art that it is difficult to efficiently, accurately, and sensitively detect multiple markers of five poultry species in a single test.

[0005] In a first aspect, this application provides an MNP marker site for bird identification, wherein the MNP marker site is a species-specific genomic region selected on each bird genome and having multiple nucleotide polymorphisms within the species; the MNP marker site includes at least one of 15 marker sites from MNP-1 to MNP-15.

[0006] Furthermore, the poultry includes at least one of chicken, duck, goose, pigeon and quail.

[0007] In a second aspect, this application provides a multiplex PCR primer composition for detecting the MNP marker site described in the first aspect, the multiplex PCR primer composition comprising at least one of a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, a tenth primer pair, an eleventh primer pair, a twelfth primer pair, a thirteenth primer pair, a fourteenth primer pair, and a fifteenth primer pair;

[0008] The upstream primer of the first primer pair is shown in SEQ ID NO.1, and the downstream primer of the first primer pair is shown in SEQ ID NO.2;

[0009] The upstream primer of the second primer pair is shown in SEQ ID NO.3, and the downstream primer of the second primer pair is shown in SEQ ID NO.4;

[0010] The upstream primer of the third primer pair is shown in SEQ ID NO.5, and the downstream primer of the third primer pair is shown in SEQ ID NO.6;

[0011] The upstream primer of the fourth primer pair is shown in SEQ ID NO.7, and the downstream primer of the fourth primer pair is shown in SEQ ID NO.8;

[0012] The upstream primer of the fifth primer pair is shown in SEQ ID NO.9, and the downstream primer of the fifth primer pair is shown in SEQ ID NO.10;

[0013] The upstream primer of the sixth primer pair is shown in SEQ ID NO.11, and the downstream primer of the sixth primer pair is shown in SEQ ID NO.12;

[0014] The upstream primer of the seventh primer pair is shown in SEQ ID NO.13, and the downstream primer of the seventh primer pair is shown in SEQ ID NO.14;

[0015] The upstream primer of the eighth primer pair is shown in SEQ ID NO.15, and the downstream primer of the eighth primer pair is shown in SEQ ID NO.16;

[0016] The upstream primer of the ninth primer pair is shown in SEQ ID NO.17, and the downstream primer of the ninth primer pair is shown in SEQ ID NO.18;

[0017] The upstream primer of the tenth primer pair is shown in SEQ ID NO.19, and the downstream primer of the tenth primer pair is shown in SEQ ID NO.20;

[0018] The upstream primer of the eleventh primer pair is shown in SEQ ID NO.21, and the downstream primer of the eleventh primer pair is shown in SEQ ID NO.22;

[0019] The upstream primer of the twelfth primer pair is shown in SEQ ID NO.23, and the downstream primer of the twelfth primer pair is shown in SEQ ID NO.24;

[0020] The upstream primer of the thirteenth primer pair is shown in SEQ ID NO.25, and the downstream primer of the thirteenth primer pair is shown in SEQ ID NO.26;

[0021] The upstream primer of the fourteenth primer pair is shown in SEQ ID NO.27, and the downstream primer of the fourteenth primer pair is shown in SEQ ID NO.28;

[0022] The upstream primer of the fifteenth primer pair is shown in SEQ ID NO.29, and the downstream primer of the fifteenth primer pair is shown in SEQ ID NO.30.

[0023] Thirdly, this application provides a reagent for detecting the MNP marker site described in the first aspect, the reagent comprising the multiplex PCR primer composition described in the second aspect.

[0024] Fourthly, this application provides a kit for detecting the MNP marker sites described in the first aspect, the kit comprising the multiplex PCR primer composition described in the second aspect or the reagents described in the third aspect.

[0025] Furthermore, the kit also includes a multiplex PCR premix.

[0026] Fifthly, this application provides the application of the MNP marker site described in the first aspect, the multiplex PCR primer composition described in the second aspect, or the reagent described in the third aspect in the preparation of a detection reagent for identifying and distinguishing meat or meat products of five kinds of poultry.

[0027] Sixthly, this application provides the application of the MNP marker site described in the first aspect, or the multiplex PCR primer composition described in the second aspect, or the reagent described in the third aspect, or the kit described in the fourth aspect, in identifying and distinguishing meat or meat products from five types of poultry.

[0028] The technical solutions provided in this application have the following advantages compared with the prior art:

[0029] This application provides an MNP marker site for poultry identification. By analyzing the genome sequences of five poultry species, a total of 15 MNP marker sites were screened. Based on the sequence information of these 15 MNP marker sites, a multiplex primer composition was designed. Multiplex PCR amplification was performed using the designed multiplex primer composition, and the amplified products were sequenced using a next-generation sequencing platform. This allows for high-throughput, high-efficiency, and high-accuracy detection and differentiation of five poultry species and their meat products in a single operation. It enables accurate identification and differentiation of the five poultry meat products, thus providing technical support for poultry identification and adulteration identification and traceability analysis of their meat products. Attached Figure Description

[0030] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0031] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a schematic diagram illustrating the principle of MNP marker polymorphism provided in an embodiment of this application;

[0033] Figure 2 A flowchart illustrating the screening and primer design process for five avian MNP marker sites provided in this application embodiment;

[0034] Figure 3 This is a flowchart illustrating the detection process of MNP marker sites provided in an embodiment of this application. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0036] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0037] This application provides an MNP marker site for bird identification, wherein the MNP marker site is a species-specific genomic region selected on each bird genome and having multiple nucleotide polymorphisms within the species; the MNP marker site includes at least one of 15 marker sites from MNP-1 to MNP-15.

[0038] In some alternative implementations, the poultry includes at least one of chicken, duck, goose, pigeon, and quail.

[0039] In this embodiment of the application, the specific types of poultry genomes are limited to five types of poultry. The resulting multiple primer composition for MNP marker sites can be amplified in samples of the five types of poultry, ensuring high-throughput, high-efficiency, and high-accuracy detection of the five types of poultry and their meat products.

[0040] Based on a general inventive concept, embodiments of this application provide a multiplex PCR primer composition for detecting the MNP marker site, the multiplex PCR primer composition comprising at least one of a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, a tenth primer pair, an eleventh primer pair, a twelfth primer pair, a thirteenth primer pair, a fourteenth primer pair, and a fifteenth primer pair;

[0041] The upstream primer of the first primer pair is shown in SEQ ID NO.1, and the downstream primer of the first primer pair is shown in SEQ ID NO.2;

[0042] The upstream primer of the second primer pair is shown in SEQ ID NO.3, and the downstream primer of the second primer pair is shown in SEQ ID NO.4;

[0043] The upstream primer of the third primer pair is shown in SEQ ID NO.5, and the downstream primer of the third primer pair is shown in SEQ ID NO.6;

[0044] The upstream primer of the fourth primer pair is shown in SEQ ID NO.7, and the downstream primer of the fourth primer pair is shown in SEQ ID NO.8;

[0045] The upstream primer of the fifth primer pair is shown in SEQ ID NO.9, and the downstream primer of the fifth primer pair is shown in SEQ ID NO.10;

[0046] The upstream primer of the sixth primer pair is shown in SEQ ID NO.11, and the downstream primer of the sixth primer pair is shown in SEQ ID NO.12;

[0047] The upstream primer of the seventh primer pair is shown in SEQ ID NO.13, and the downstream primer of the seventh primer pair is shown in SEQ ID NO.14;

[0048] The upstream primer of the eighth primer pair is shown in SEQ ID NO.15, and the downstream primer of the eighth primer pair is shown in SEQ ID NO.16;

[0049] The upstream primer of the ninth primer pair is shown in SEQ ID NO.17, and the downstream primer of the ninth primer pair is shown in SEQ ID NO.18;

[0050] The upstream primer of the tenth primer pair is shown in SEQ ID NO.19, and the downstream primer of the tenth primer pair is shown in SEQ ID NO.20;

[0051] The upstream primer of the eleventh primer pair is shown in SEQ ID NO.21, and the downstream primer of the eleventh primer pair is shown in SEQ ID NO.22;

[0052] The upstream primer of the twelfth primer pair is shown in SEQ ID NO.23, and the downstream primer of the twelfth primer pair is shown in SEQ ID NO.24;

[0053] The upstream primer of the thirteenth primer pair is shown in SEQ ID NO.25, and the downstream primer of the thirteenth primer pair is shown in SEQ ID NO.26;

[0054] The upstream primer of the fourteenth primer pair is shown in SEQ ID NO.27, and the downstream primer of the fourteenth primer pair is shown in SEQ ID NO.28;

[0055] The upstream primer of the fifteenth primer pair is shown in SEQ ID NO.29, and the downstream primer of the fifteenth primer pair is shown in SEQ ID NO.30.

[0056] In this embodiment, the composition of the specific multiplex amplification primer composition for detecting MNP marker sites is refined, enabling full coverage detection of five poultry species, thereby achieving high-throughput, high-efficiency, and high-accuracy detection of the five poultry species and their meat products.

[0057] The multiplex PCR primer composition is designed based on the above-mentioned MNP marker site. The specific composition and region of the MNP marker site can be referred to the above embodiments. Since the multiplex PCR primer composition adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0058] Based on a general inventive concept, embodiments of this application provide a reagent for detecting the MNP marker site, the reagent comprising the multiplex PCR primer composition.

[0059] This reagent is based on the above-described multiplex PCR primer composition. The specific sequence information of the multiplex PCR primer composition can be found in the above embodiments. Since this reagent adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0060] Based on a general inventive concept, embodiments of this application provide a kit for detecting the MNP marker site, the kit comprising the multiplex PCR primer composition or the reagent.

[0061] This kit is based on the above-described multiplex PCR primer composition. The specific sequence information of the multiplex PCR primer composition can be found in the above embodiments. Since this kit adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0062] Based on a general inventive concept, embodiments of this application provide the application of the MNP marker site, the primer composition, or the reagent in the preparation of a detection reagent for identifying and distinguishing meat or meat products from five types of poultry.

[0063] This application is based on the above-mentioned MNP marker sites. The specific composition and region of the MNP marker sites can be referred to in the above embodiments. Since this application adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0064] Based on a general inventive concept, embodiments of this application provide the application of the MNP marker site, the primer composition, the reagent, or the kit in identifying and distinguishing meat or meat products from five types of poultry.

[0065] In this embodiment of the application, when used for the identification of five types of poultry and their meat products, the number of sequencing sequences and the number of MNP markers detected for each type of poultry in the test sample and blank control are used to determine whether the test sample contains the nucleic acid of that poultry. The quality control scheme and determination method are based on known poultry DNA as the test sample, to evaluate the efficiency and accuracy of the kit in detecting each type of poultry, and to formulate the quality control scheme and determination method for the kit to detect five types of poultry.

[0066] This application is based on the above-mentioned MNP marker sites. The specific composition and region of the MNP marker sites can be referred to in the above embodiments. Since this application adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0067] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. If there is no corresponding national standard, then general international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0068] The creative thinking behind this application is:

[0069] The novel avian-specific molecular markers developed in this application are MNP marker sites. MNP marker sites refer to polymorphic markers caused by multiple nucleotides in a region of the genome. Compared with SSR markers and SNP markers, MNP markers have the following advantages:

[0070] (1) Abundant alleles, with 2 alleles on a single MNP marker. n Species alleles, higher than SSR and SNP, are suitable for the identification and differentiation of organisms;

[0071] (2) It has strong species differentiation ability and only requires a small number of MNP markers to achieve species identification, thus reducing the detection error rate.

[0072] The method described in this application is based on ultramultiplex PCR combined with next-generation high-throughput sequencing technology, and can detect 15 MNP markers from 5 avian species in a single test. It has the following advantages:

[0073] (1) High efficiency: Multiplex PCR is used to detect 15 markers of 5 poultry species at once, while existing fluorescent PCR technology can detect 1 marker of one poultry species at once, which increases the detection efficiency by 15 times.

[0074] (2) High accuracy: Multiplex PCR is used to detect multiple markers in multiple birds at once, avoiding high false negatives caused by the failure of amplification of a single marker; the amplification products are sequenced hundreds of times using a second-generation high-throughput sequencer, and the detection results are accurate and reliable.

[0075] (3) Detection and separation of mixtures: 15 MNP markers of 5 poultry species are detected at one time. Based on the detected MNP markers, the poultry species in the mixture are separated. Existing methods based on fluorescent PCR technology are limited by the number of fluorescent channels and background fluorescence signals, making it difficult to detect multiple markers of multiple poultry species at the same time.

[0076] Given the above advantages and characteristics, MNP labeling and its detection technology: MNP labeling can identify and distinguish cross-species mixtures, and has application potential in meat adulteration and traceability. Currently, there are no reports on MNP labeling in the detection of poultry meat products, and corresponding technologies are lacking.

[0077] Based on this, this application developed MNP marker sites for five bird species. The MNP marker sites are genomic regions specific to each bird species and having multiple nucleotide polymorphisms within the species, screened on the genomes of the five bird species. The reference genome for each MNP marker site is shown in Table X.

[0078] This application develops a multiplex PCR primer composition for the five avian MNP marker sites. The multiplex PCR primer composition includes 15 primer pairs, and the nucleotide sequences of the 15 primer pairs are shown in SEQ ID NO.1 to SEQ ID NO.30. The primers do not conflict with each other and can be efficiently amplified by multiplex PCR.

[0079] The multiplex PCR primer composition can be used as a detection kit for the five MNP marker sites in poultry, and the kit provided in this application can accurately identify and distinguish the five poultry species.

[0080] In the reproducibility test of this application, the logarithm of the difference in major MNP marker genotypes between different libraries and between different library construction batches for each sample was 0, the reproducibility r = 100%, and the accuracy a = 100%.

[0081] The MNP markers and the kit described in this application have high specificity for detecting each type of poultry.

[0082] Example 1

[0083] Screening of 5 avian MNP marker sites and design of multiplex PCR amplification primers:

[0084] Screening of 5 MNP marker loci in birds:

[0085] like Figure 1 As shown, based on the publicly available genome sequences of each of the five bird species, five MNP marker sites were selected for each bird species through sequence alignment, for a total of 15 MNP marker sites. The 15 selected MNP marker sites are shown in Table 1.

[0086] Table 1. MNP marker sites and the starting positions of multiplex PCR amplification primers on the reference sequence.

[0087]

[0088]

[0089] The specific steps for the above screening are as follows:

[0090] The genome sequence of a representative strain of each type of bird is selected as a reference genome, and the other genome sequences of the bird are compared with the reference genome to obtain the single nucleic acid polymorphism sites of the bird.

[0091] On the reference genome, a window of 100bp to 300bp is used for window translation with a step size of 1bp to screen and obtain regions of multiple candidate MNP marker sites. The candidate MNP marker site regions are required to contain ≥2 of the single nucleotide variant sites and there are no single nucleotide polymorphism sites on the sequences at both ends of 30bp.

[0092] At least five sites with high DP (discrimination) values ​​in the candidate polynucleotide polymorphism (PNP) regions were selected; the selection criteria included the following:

[0093] DP = d / t,

[0094] In the formula, t is the number of comparisons when all genomes are compared pairwise in the candidate polynucleotide polymorphism site region, and d is the number of genome pairs with at least two single nucleotide polymorphism differences in the candidate polynucleotide polymorphism site region.

[0095] Other step sizes can also be used in the window translation stage, not limited to 1bp, but the use of 1bp in this application is beneficial for comprehensive screening of 5 poultry species.

[0096] After obtaining the MNP marker sites for each type of poultry, sequence alignment was performed in a public database, and at least 5 poultry-specific sites were selected as candidate MNP marker sites for each type of poultry.

[0097] Design of primers for multiplex PCR amplification:

[0098] like Figure 2 As shown, multiplex PCR amplification primers for the candidate MNP marker were designed using primer design software. The primer design followed the principle that primers do not interfere with each other, and all primers can be combined into a primer pool for multiplex PCR amplification, that is, all designed primers can be amplified normally in one amplification reaction.

[0099] Evaluation of the detection efficiency of primer combinations:

[0100] Positive samples of each type of poultry purchased from the market were mixed with equal copy numbers after detecting copy numbers using digital PCR, thus preparing a mixed sample containing the five types of poultry. The MNP marker detection kit was used to detect single and mixed samples of each type of poultry, totaling eight samples. Each single sample was tested once, and the mixed sample was tested in triplicate. Sequencing data were analyzed, and based on the sites detected in the single and mixed samples, a set of MNP marker sites and compatible primer combinations were selected. Five MNP markers were selected for each type of poultry.

[0101] Example 2

[0102] Comparing Example 2 with Example 1, the difference between Example 2 and Example 1 is as follows:

[0103] Performance evaluation and threshold setting for MNP marker and primer identification of each of the five bird species:

[0104] Five poultry species were prepared as a mixed sample, with an equal volume of sterile water as a blank control. Three replicate libraries were analyzed for each sample over four consecutive days, resulting in 12 sequencing data sets per sample. Based on the data analysis results for each sample shown in Table 2, the reproducibility and accuracy of the detection method were evaluated, and thresholds for contamination in the quality control system and detection of target poultry species were established. The detection procedure for MNP marker sites is as follows: Figure 3 As shown.

[0105] Table 2 shows the sensitivity and stability analysis of the MNP marker detection kit for each of the five poultry species.

[0106]

[0107] As shown in Table 2, the kit can stably detect all MNP markers in the mixed sample for 4 consecutive days, indicating that the MNP marker detection kit has detection sensitivity and technical stability for each type of poultry.

[0108] During the experiment, it was found that all five MNP marker sites of each bird species were detected in the mixed sample, and the sequences specifically aligned with the reference sequences of the respective birds, indicating the high specificity of the primers and detection method for detecting each bird species. A single reaction detected five bird species in the sample, with five markers detected for each species, demonstrating the high efficiency and accuracy of the primers and detection method in detecting and separating the mixed bird species.

[0109] Evaluation of the reproducibility and accuracy of the MNP marker detection kit in five avian species:

[0110] Based on whether the genotypes of the common detection sites in the two replicates can be reproduced, the reproducibility and accuracy of the MNP marker detection method in five poultry species were evaluated. Specifically, the genotypes of each MNP site generated from the 12 sets of data of the mixed samples were compared pairwise, and the results are shown in Table 3.

[0111] Table 3. Reproducibility and accuracy assessment of MNP marker detection kit for genotype detection

[0112] Repeat 1 Repeat 2 Number of common sites Number of repeatable sites Recurrence rate r Accuracy a S-1 S-2 15 15 100％ 100％ S-1 S-3 15 15 100％ 100％ S-1 S-4 15 15 100％ 100％ S-1 S-5 15 15 100％ 100％ S-1 S-6 15 15 100％ 100％ S-1 S-7 15 15 100％ 100％ S-1 S-8 15 15 100％ 100％ S-1 S-9 15 15 100％ 100％ S-1 S-10 15 15 100％ 100％ S-1 S-11 15 15 100％ 100％ S-1 S-12 15 15 100％ 100％

[0113] As shown in Table 3, the number of MNP markers with different major genotypes was 0. Based on the principle that reproducible genotypes between two replicates are considered accurate, the accuracy a = 1 - (1 - r) / 2 = 0.5 + 0.5r, where r represents the reproducibility rate, i.e., the ratio of the number of reproducible loci to the number of shared loci. In the reproducibility test of this invention, the logarithm of the difference in major genotypes of MNP markers between different libraries and between different library preparation batches for each sample was 0, the reproducibility rate r = 100%, and the accuracy a = 100%.

[0114] In the mixed sample, five MNP markers could be reliably detected for each type of poultry, while a maximum of one MNP marker was detected in the blank control. Therefore, the criterion for determining each positive result in this application is: when no less than two MNP marker sites are detected for each type of poultry in the sample, the nucleic acid of the poultry is determined to be detected in the sample.

[0115] Example 3

[0116] Comparing Example 3 and Example 2, the differences between Example 3 and Example 2 are as follows:

[0117] Application of MNP marker sites, kits, and methods in the identification and differentiation of five commercially available poultry meat products:

[0118] Artificially prepared minced meat products were tested using an MNP marker site detection kit. The samples were named S1-S9 in sequence, and poultry in the samples were identified. The results are shown in Table 4.

[0119] Table 4. Analysis of the detection results of 9 out of 5 types of poultry meat products

[0120]

[0121] As shown in Table 4, the kit and method detected five MNP marker sites (S1-S5) in meat products containing one of the poultry species in a single test, accurately identifying the expected poultry species. In contrast, a single-reaction method based on fluorescent PCR to detect one marker requires five tests, demonstrating the accuracy and efficiency of the kit and method in detecting single poultry meat products.

[0122] As shown in Table 4, the kit and method described herein can accurately separate mixtures of meat products from multiple poultry sources in a single reaction. In contrast, the method based on fluorescent PCR, which detects one label in a single reaction, requires 15 tests to achieve the desired detection effect. This demonstrates the high efficiency and accuracy of the kit and method described herein in detecting meat adulteration.

[0123] One or more technical solutions in the embodiments of this application have at least the following technical effects or advantages:

[0124] (1) The 15 highly polymorphic MNP marker sites provided in this application have high specificity in identifying and distinguishing 5 kinds of birds.

[0125] (2) The high polymorphism multiplex PCR amplification primer composition provided in this application can amplify 15 MNP marker sites in one reaction and integrate with a second-generation sequencing platform to sequence the amplification products, so as to realize the common detection and differentiation of 5 poultry species in one reaction, and provide technical support for the efficient and accurate identification and differentiation of 5 poultry species and their meat products.

[0126] (3) The kit for high polymorphic MNP marker sites provided in this application can detect all 15 MNP marker sites in a single reaction. In the reproducibility test, all 15 marker sites were stably detected, demonstrating the high stability of the kit. The logarithm of the difference in major MNP marker genotypes between different libraries and different batches of libraries detected by the kit was 0, with a reproducibility rate r = 100% and an accuracy a = 100%, indicating the high accuracy and stability of the kit in complex templates.

[0127] (4) The primer composition provided in this application embodiment can be used to detect samples by high-throughput sequencing. By adding a unique tag to each sample, it is possible to detect thousands of samples at once, which can further improve the detection efficiency.

[0128] (5) The application provided in this application, compared with the traditional fluorescent PCR method which relies on parallel experiments of standard samples to detect fluorescent signals, only requires high-throughput sequencing to detect the sample, and then identification of poultry by the detected MNP-labeled base sequence. Parallel detection of standard samples is not required, the operation is simple and the data processing is simple, which improves the detection efficiency.

[0129] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0130] In this application, unless otherwise stated, directional terms such as "upper" and "lower" specifically refer to the orientation shown in the accompanying drawings. Furthermore, in the description of this application, the terms "comprising," "including," etc., mean "including but not limited to."

[0131] In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, without necessarily requiring or implying any actual relationship or order between these entities or operations. In this document, "and / or" describes the association between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. A and B can be singular or plural. In this document, "at least one" means one or more, and "more than" means two or more. "At least one," "at least one of the following," or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c," or "at least one of a, b, and c," can both represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0132] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A multiplex PCR primer composition for detecting MNP marker sites for avian identification, characterized in that, The MNP marker sites are species-specific genomic regions selected from the genomes of each type of poultry, which have multiple nucleotide polymorphisms within the species; the poultry are chickens, ducks, geese, pigeons, and quails; The multiplex PCR primer composition is a combination of the first primer pair, the second primer pair, the third primer pair, the fourth primer pair, the fifth primer pair, the sixth primer pair, the seventh primer pair, the eighth primer pair, the ninth primer pair, the tenth primer pair, the eleventh primer pair, the twelfth primer pair, the thirteenth primer pair, the fourteenth primer pair, and the fifteenth primer pair; The upstream primer of the first primer pair is shown in SEQ ID NO.1, and the downstream primer of the first primer pair is shown in SEQ ID NO.2; The upstream primer of the second primer pair is shown in SEQ ID NO.3, and the downstream primer of the second primer pair is shown in SEQ ID NO.4; The upstream primer of the third primer pair is shown in SEQ ID NO.5, and the downstream primer of the third primer pair is shown in SEQ ID NO.6; The upstream primer of the fourth primer pair is shown in SEQ ID NO.7, and the downstream primer of the fourth primer pair is shown in SEQ ID NO.8; The upstream primer of the fifth primer pair is shown in SEQ ID NO.9, and the downstream primer of the fifth primer pair is shown in SEQ ID NO.10; The upstream primer of the sixth primer pair is shown in SEQ ID NO.11, and the downstream primer of the sixth primer pair is shown in SEQ ID NO.12; The upstream primer of the seventh primer pair is shown in SEQ ID NO.13, and the downstream primer of the seventh primer pair is shown in SEQ ID NO.14; The upstream primer of the eighth primer pair is shown in SEQ ID NO.15, and the downstream primer of the eighth primer pair is shown in SEQ ID NO.16; The upstream primer of the ninth primer pair is shown in SEQ ID NO.17, and the downstream primer of the ninth primer pair is shown in SEQ ID NO.18; The upstream primer of the tenth primer pair is shown in SEQ ID NO.19, and the downstream primer of the tenth primer pair is shown in SEQ ID NO.20; The upstream primer of the eleventh primer pair is shown in SEQ ID NO.21, and the downstream primer of the eleventh primer pair is shown in SEQ ID NO.22; The upstream primer of the twelfth primer pair is shown in SEQ ID NO.23, and the downstream primer of the twelfth primer pair is shown in SEQ ID NO.24; The upstream primer of the thirteenth primer pair is shown in SEQ ID NO.25, and the downstream primer of the thirteenth primer pair is shown in SEQ ID NO.26; The upstream primer of the fourteenth primer pair is shown in SEQ ID NO.27, and the downstream primer of the fourteenth primer pair is shown in SEQ ID NO.28; The upstream primer of the fifteenth primer pair is shown in SEQ ID NO.29, and the downstream primer of the fifteenth primer pair is shown in SEQ ID NO.

30.

2. A reagent for detecting MNP marker sites for identification in poultry, characterized in that, The reagent includes the multiplex PCR primer composition as described in claim 1.

3. A kit for detecting MNP marker sites for identification in poultry, characterized in that, The kit includes the multiplex PCR primer composition as described in claim 1 or the reagent as described in claim 2.

4. The reagent kit according to claim 3, characterized in that, The kit also includes a multiplex PCR premix.

5. The use of a multiplex PCR primer composition as described in claim 1 or a reagent as described in claim 2 in the preparation of a detection reagent for identifying and distinguishing meat from five types of poultry, wherein the poultry are chicken, duck, goose, pigeon and quail.

6. The use of a multiplex PCR primer composition as described in claim 1, or a reagent as described in claim 2, or a kit as described in claim 3 or 4, in identifying and distinguishing meat from five types of poultry, namely chicken, duck, goose, pigeon, and quail.

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