WO2020238218A1 - Combinaison de marqueur snp et procédé d'identification pour porc noir jiaxing et un produit carné cru - Google Patents

Combinaison de marqueur snp et procédé d'identification pour porc noir jiaxing et un produit carné cru Download PDF

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WO2020238218A1
WO2020238218A1 PCT/CN2019/130974 CN2019130974W WO2020238218A1 WO 2020238218 A1 WO2020238218 A1 WO 2020238218A1 CN 2019130974 W CN2019130974 W CN 2019130974W WO 2020238218 A1 WO2020238218 A1 WO 2020238218A1
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pig
pig13
identification
pig2
site
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PCT/CN2019/130974
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English (en)
Chinese (zh)
Inventor
王起山
潘玉春
张哲�
王振
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浙江大学
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Publication of WO2020238218A1 publication Critical patent/WO2020238218A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to the field of food safety monitoring, in particular to a SNP mark combination and identification method of Jiaxing black pig and raw meat products.
  • the main local breeds of pigs in the Taihu Lake Basin are: Erhualian pigs, Meishan pigs, Fengjing pigs, Shawutou pigs, rice pigs, and Jiaxing black pigs.
  • Meishan pigs are divided into Zhongmeishan pigs and Xiaomeishan pigs due to their body size.
  • the local breeds of pigs in the Taihu Basin share the same roots and have their own characteristics. However, due to their similar body shapes, especially piglets, there is confusion in the production.
  • the traditional body shape and appearance breed identification methods are used to identify the various breeds of the local pigs in the Taihu Basin.
  • Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level, which has abundant sites, wide distribution, high genetic stability, It is representative, convenient and quick to detect. Among them, within a certain breed (group), SNPs that only appear in one group are called breed-specific SNPs. Variety-specific SNP site screening and site combination method research is the key to the identification of varieties and products.
  • the third-generation molecular marker SNP Compared with the previous two generations of molecular markers, the third-generation molecular marker SNP has the advantages of rich variation, low requirements for DNA samples, high stability, accurate determination, simple detection methods and high throughput. At present, the third-generation molecular marker SNP has been widely used in the fields of paternity identification, animal and plant species (line) identification, genetic breeding and other fields.
  • the purpose of the present invention is to address the shortcomings of the prior art and propose a SNP marker combination and identification method for identifying Jiaxing black pigs and raw meat products, using third-generation molecular marker identification and Sanger sequencing technology to identify Jiaxing black pigs and raw meat products.
  • a SNP marker combination and identification method for identifying Jiaxing black pigs and raw meat products, using third-generation molecular marker identification and Sanger sequencing technology to identify Jiaxing black pigs and raw meat products.
  • a combination of SNP markers for Jiaxing black pig and raw meat products including: pig2-60239356, pig8-5778954, pig7-37079960, pig13-16955200 and pig13-111071937; said pig2-60239356 represents the 60239356th locus of pig chromosome 2 , Pig8-5778954 represents the 5778954 locus of pig chromosome 8, pig7-37079960 represents the 37079960 position of pig chromosome 7, pig13-16955200 represents the 16955200 position of pig chromosome 13, and pig13-111071937 represents pig 13 Chromosome 111071937 position.
  • the Jiaxing black pig and its raw meat product identification method based on the combination of SNP markers first extract the genomic DNA of the raw pork or meat product to be identified, and then perform agarose gel electrophoresis and Sanger sequencing after PCR amplification to obtain SNPs The information of each detection site of the tag combination, when a specific mutation occurs at any three sites of the SNP tag combination, it can be identified as Jiaxing black pig and its meat products.
  • the sequences of the upstream and downstream primers at pig2-60239356 are shown in SEQ ID Nos. 1 to 2, and the sequences of the upstream and downstream primers at pig8-5778954 are shown in SEQ ID Nos. 3 to 4.
  • the sequences of the upstream and downstream primers at pig7-37079960 are shown in SEQ ID Nos. 5-6
  • the sequences of the upstream and downstream primers at pig13-16955200 are shown in SEQ ID Nos. 7-8
  • the upper and lower primers at pig13-111071937 The sequence of the downstream primer is shown in SEQ ID NO. 9-10.
  • comparison information of identification sites of sequencing products is shown in the following table:
  • REF represents the reference genotype
  • ALT represents the mutant genotype
  • the identification site information is: the mutation genotype and the reference genotype of each detection site.
  • the site is considered to have no identification significance.
  • the locus is considered to be of identification significance.
  • the beneficial effect of the present invention is that compared with the prior art, the present invention uses the unique SNP sites of Jiaxing black pig breeds as the identification basis, studies the method for identifying Jiaxing black pigs from the molecular level, and uses Sanger sequencing as the main molecular identification method.
  • the present invention relates to a SNP marker of Jiaxing black pig and raw meat products, including: pig2-60239356, pig8-5778954, pig7-37079960, pig13-16955200 and pig13-111071937; said pig2-60239356 represents the 60239356th position of pig chromosome 2 Pig8-5778954 represents the 5778954 locus of pig chromosome 8, pig7-37079960 represents the 37799960 position of pig chromosome 7, pig13-16955200 represents the 16955200 position of pig chromosome 13, and pig13-111071937 represents pig Position 111071937 on chromosome 13.
  • the present invention provides a method for identifying Jiaxing black pigs and raw meat products based on the above-mentioned SNP marker combination.
  • the genomic DNA of raw pork or meat products to be identified is first extracted, and then amplified by PCR and then subjected to agarose gel electrophoresis and Sanger sequencing , Get the information of each detection site of the SNP marker combination.
  • a specific mutation occurs at any three sites of the SNP marker combination, it can be identified as Jiaxing black pig and its meat products.
  • F represents the upstream primer
  • R represents the downstream primer
  • length represents the length of the standard product
  • F'-position represents the position of the SNP site in the amplified product.
  • the reaction system is template DNA 1ng/uL, primer 1uL, H 2 O 3.8uL, 2 ⁇ Taq PCR Masrer Mix 50uL; and/or, the PCR reaction program is 95°C pre-denaturation 2min , 95°C pre-denaturation 30s, 60°C annealing temperature 30s, 72°C extension 1min, cycle times 30 times, 72°C extension 10min.
  • the mass concentration of the agarose gel is 2%.
  • the band length requirements are as shown in Table 1.
  • REF represents the reference genotype
  • ALT represents the mutant genotype
  • the mutant genotypes and reference genotypes of each detection site When the reference genotype appears at the test site of the pig to be tested, the site is considered to be of no identification significance. For example, if a pig A to be tested is located at pig2-60239356, if the sequencing data is C, it is considered that the pig A to be tested has no identification significance at pig2-60239356; if the sequencing data is T, it is considered that pig A to be tested has identification significance at pig2-60239356.
  • the present invention uses a combination of sites as an identification Marker.
  • Marker1-10 are the identification marker combinations for Jiaxing black pigs. For example, when pig A to be tested has any one of the 10 Marker combinations, then pig A to be tested is considered Jiaxing black pig. .
  • the Jiaxing black pork products refer to Jiaxing black pig cut meat and pickled products and cooked food products processed and prepared using Jiaxing black pig as raw materials.
  • the primers are used for PCR amplification of DNA samples.
  • the reaction system of the PCR reaction is a 10uL system: template DNA 1ng/uL, primers 1uL, H 2 O 3.8uL, 2 ⁇ Taq PCR Masrer Mix 50uL; and/or, the PCR reaction program is 95°C pre-denaturation 2min, 95°C Pre-denaturation for 30 seconds, annealing temperature at 60°C for 30 seconds, extension at 72°C for 1 min, 30 cycles, and extension at 72°C for 10 min.
  • the gel electrophoresis conditions are current 10A, voltage 100V, time 40 minutes.
  • the amplified products of different primers are compared with the standard product length in Table 1. If the product length is within the error range and consistent with the standard product length, the amplification result is considered qualified.
  • REF represents the reference genotype
  • ALT represents the mutant genotype
  • ⁇ represents the detected mutant genotype
  • Pig No. 1 detected mutations at pig2-60239356, pig8-5778954, pig7-37079960, pig13-16955200, and pig13-111071937, which are in line with Marker1, Marker2, Marker3, Marker4, Marker5
  • the identification information of Marker6, Marker7, Marker8, Marker9, Marker10 identified pig No. 1 as Jiaxing black pig; pig No. 2 had no mutation detected and did not meet the Marker information, and pig No.
  • the identification method uses the third-generation molecular marker SNP to overcome the shortcomings of fewer available sites for the first two generations of molecular markers, and simplifies the identification method compared with the identification technology of the first two generations of molecular markers.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une combinaison de marqueurs de SNP et un procédé d'identification pour un porc noir Jiaxing et un produit carné cru. Le procédé comprend : l'extraction D'un ADN génomique de viande crue brute ou d'un produit carné, la réalisation d'une amplification par PCR, puis la réalisation d'une électrophorèse sur gel d'agarose et d'un séquençage de Sanger, et l'identification d'un porc noir Jiaxing et d'un produit carné correspondant selon un génotype SNP d'un site particulier dans le résultat de séquençage, les sites d'identification du séquençage de Sanger étant les sites pig2-60239356, pig8-5778954, pig7-37079960, pig13-16955200, et pig13-111071937 où des mutations spécifiques se produisent.
PCT/CN2019/130974 2019-05-30 2019-12-31 Combinaison de marqueur snp et procédé d'identification pour porc noir jiaxing et un produit carné cru WO2020238218A1 (fr)

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CN201910462678.4A CN110106261B (zh) 2019-05-30 2019-05-30 嘉兴黑猪及生肉制品的snp标记组合和鉴定方法

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CN110106261B (zh) * 2019-05-30 2020-11-24 浙江大学 嘉兴黑猪及生肉制品的snp标记组合和鉴定方法
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