WO2020238217A1 - 浦东白猪及生肉制品的snp标记组合和鉴定方法 - Google Patents

浦东白猪及生肉制品的snp标记组合和鉴定方法 Download PDF

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WO2020238217A1
WO2020238217A1 PCT/CN2019/130973 CN2019130973W WO2020238217A1 WO 2020238217 A1 WO2020238217 A1 WO 2020238217A1 CN 2019130973 W CN2019130973 W CN 2019130973W WO 2020238217 A1 WO2020238217 A1 WO 2020238217A1
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pig
pudong
white
identification
meat products
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潘玉春
王起山
张哲�
王振
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浙江大学
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Priority to US17/042,122 priority Critical patent/US20220228225A1/en
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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
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • 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

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  • the invention relates to the field of food safety monitoring, in particular to a SNP mark combination and identification method for Pudong white pigs and raw meat products.
  • Pudong white pigs are mainly distributed in Shanghai Nanhui and Chuansha. It is characterized by high reproduction rate and early sexual maturity, with an average of about 12 litters per litter. After castration, it is quiet and less moving, suitable for soft-circle breeding. Before the 1950s, it was the master breed in Chuansha County. Because of its delicious meat, it is called "glutinous pig" and is popular among consumers. Pudong white pigs are similar in appearance to Western Landrace pigs and large white pigs, and Western pig breeds are used to pretend to be Pudong white pigs in production.
  • the distribution area of Pudong white pigs overlaps with the distribution areas of Taihu pig breeds (Erhualian pig, Meishan pig, Fengjing pig, Shawutou pig, rice pig, Jiaxing black pig), and there is confusion in production. It is very difficult to distinguish Pudong white pigs from other breeds of pigs by traditional methods, especially the cut pigs after slaughter and their processed meat products. With the development of sequencing technology, molecular markers have also developed from the first generation of restriction fragment length polymorphism (RFLP), the second generation of variable number tandem repeat polymorphism (SSR), to single nucleotide polymorphism ( SNP).
  • RFLP restriction fragment length polymorphism
  • SSR variable number tandem repeat polymorphism
  • SNP single nucleotide polymorphism
  • 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 Pudong white pigs and raw meat products, using third-generation molecular marker identification and Sanger sequencing technology to identify Pudong white pigs and raw meat products, and solve the existing problems
  • a combination of SNP markers for Pudong white pigs and raw meat products including: pig18-52722267, pig8-146130825, pig9-10041850 and pig13-213464983, said pig18-52722267 represents the 52722267 locus of pig chromosome 18, and pig8-146130825 represents Pig chromosome 8 is at position 146130825, pig9-10041850 is at position 10041850 on pig chromosome 9, and pig13-213464983 is at position 213464983 on pig chromosome 13.
  • the Pudong white 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 the SNP marker Combining the information of each detection site, when a specific mutation occurs at any three sites of the SNP marker combination, it can be identified as Pudong white pig and its meat products.
  • sequences of the upstream and downstream primers at pig18-52722267 are shown in SEQ ID NOs. 1 and 2
  • sequences of the upstream and downstream primers at pig8-146130825 are shown in SEQ ID NOs. 3 to 4.
  • sequences of the upstream and downstream primers at pig9-10041850 are shown in SEQ ID Nos. 5-6
  • sequences of the upstream and downstream primers at pig13-213464983 are shown in SEQ ID Nos. 7-8.
  • 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 have identification significance.
  • the beneficial effects of the present invention are: compared with the prior art, the present invention uses the unique SNP sites of Pudong white pig breeds as the identification basis, studies the method for identifying Pudong white pigs from the molecular level, and uses Sanger sequencing as the main molecular identification method, which can Pudong white pigs are distinguished from common western pig breeds and local breed pigs in the Taihu Lake Basin, such as: Xiaomeishan pig, Fengjing pig, Zhongmeishan pig, Erhualian pig, Jiaxing black pig, rice pig, Shawutou pig, Landrace pig, Great White Pig, Duroc, Pietrain, Berkshire, etc.
  • the present invention relates to a SNP marker for Pudong white pigs and raw meat products, including: pig18-52722267, pig8-146130825, pig9-10041850, and pig13-213464983.
  • the pig18-52722267 represents position 52722267 of pig chromosome 18
  • pig8- 146130825 represents position 146130825 of pig chromosome 8
  • pig9-10041850 represents position 10041850 of pig chromosome 9
  • pig13-213464983 represents position 213464983 of pig chromosome 13.
  • the present invention provides a Pudong white pig and raw meat product identification method based on the above-mentioned SNP marker combination.
  • the genomic DNA of the raw pork or meat product to be identified is extracted, and then amplified by PCR and then subjected to agarose gel electrophoresis and Sanger sequencing. Obtain 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 Pudong white 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 for 2 min , 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. At this time, the site is considered to be of identification significance.
  • a pig A to be tested is at the pig18-52722267 site. If the sequencing data is T, the pig A to be tested is considered to be of no identification significance at the pig18-52722267 site; if the sequencing data is C, it is considered that pig A to be tested has identification significance at pig18-52722267 locus.
  • the present invention uses a combination of sites as an identification Marker.
  • Marker1-4 are Pudong White Pig identification marker combinations.
  • pig A to be tested has any combination of site mutations in the 4 Marker combinations, pig A to be tested is considered Pudong White pig.
  • the Pudong white pork products refer to Pudong white pig cut meat and pickled products and cooked food products prepared by processing Pudong white pigs 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.
  • Different primers compare the amplified product 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 a mutation at pig18-52722267 locus, which did not meet the Marker information, and it was identified that pig No. 1 was not a Pudong white pig; pig No. 2 was found in pig18-52722267, pig8-146130825, pig9 A mutation was detected at position -10041850, in line with the information of Marker1, and pig No. 2 was identified as Pudong white pig; pig No. 3 was detected with mutations at pig8-146130825 and pig9-10041850, which did not meet the Marker information, and pig No. 3 was identified as Pudong white pig; Pig No.
  • Pig No. 4 detected mutations at pig18-52722267 and pig13-213464983, which did not meet the Marker information.
  • Pig No. 4 was identified as Pudong white pig;
  • pig No. 5 was detected at pig8-146130825, pig9-10041850, pig13-213464983.
  • Marker4 identified pig No. 5 as Pudong white pig.

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Abstract

本发明公开了一种浦东白猪及生肉制品的SNP标记组合和鉴定方法，通过提取生猪肉或肉制品的基因组DNA，经PCR扩增后进行琼脂糖凝胶电泳和Sanger测序，根据测序结果特点位点的SNP基因型鉴定浦东白猪及其肉制品；所述的Sanger测序，其鉴定位点为：pig18-52722267、pig8-146130825、pig9-10041850和pig13-213464983位点处出现特异突变；本发明解决现有技术中尚未有浦东白猪及其肉制品相关的鉴定方法的问题。

Description

浦东白猪及生肉制品的SNP标记组合和鉴定方法 技术领域
本发明涉及的是一种食品安全监测领域，具体涉及一种浦东白猪及生肉制品的SNP标记组合和鉴定方法。
背景技术
浦东白猪主要分布在上海南汇及川沙等地。其特点是繁殖率高，性成熟早，平均每胎产仔12头左右，阉割后，安静少动，适合软圈饲养，50年代前，是川沙县的当家品种。其因肉质鲜美，称为“糯猪”而广受消费者欢迎。浦东白猪体型外貌与西方长白猪和大白猪相似，在生产中存在用西方猪种冒充浦东白猪的情况。另一方面，浦东白猪分布区域与太湖猪种(二花脸猪、梅山猪、枫泾猪、沙乌头猪、米猪、嘉兴黑猪)分布区域有重合，生产中存在混淆的情况。通过传统方法将浦东白猪与其他品种猪区分难度很大，尤其是宰杀后的分割猪及其处理后的肉制品。随着测序技术的发展，分子标记也由一代的限制性片段长度多态性(RFLP)，第二代的数目可变串联重复多态性(SSR)，发展到了单核苷酸多态性(SNP)。第三代分子标记SNP相对于前两代分子标记具有变异丰富、对DNA样本要求低、稳定性高、测定准确、检测方法简便且通量高等优点。目前，第三代分子标记SNP已广泛应用于亲子鉴定、动植物品种(品系)鉴定、遗传育种等领域。
发明内容
本发明的目的在于针对现有技术的不足，提出一种鉴定浦东白猪及生肉制品的SNP标记组合和鉴定方法，利用第三代分子标记鉴别及Sanger测序技术鉴别浦东白猪及生肉制品，解决现有技术中尚未有浦东白猪及其肉制品相关的鉴定方法的问题，提供了一种结果准确、操作简单、价格低廉的鉴定浦东白猪及其肉制品的方法和相关专用引物。
本发明的目的是通过以下技术方案实现的：
一种浦东白猪及生肉制品的SNP标记组合，包括：pig18-52722267、pig8-146130825、pig9-10041850和pig13-213464983，所述pig18-52722267表示猪18号染色体第52722267位位点，pig8-146130825表示猪8号染色体第146130825位位点，pig9-10041850表示猪9号染色体第10041850位位点，pig13-213464983表示猪13号染色体第213464983位位点。
进一步地，基于SNP标记组合的浦东白猪及其生肉制品鉴定方法，首先提取待鉴定的生猪肉或肉制品的基因组DNA，然后经PCR扩增后进行琼脂糖凝胶电泳和Sanger测序，得到SNP标记组合各个检测位点的信息，当SNP标记组合任意三个位点处出现特异突变，即可鉴 定为浦东白猪及其肉制品。
进一步地，所述的PCR扩增，pig18-52722267处的上下游引物的序列如SEQ IDNO.1～2所示，pig8-146130825处的上下游引物的序列如SEQ ID NO.3～4所示，pig9-10041850处的上下游引物的序列如SEQ ID NO.5～6所示，pig13-213464983处的上下游引物的序列如SEQ ID NO.7～8所示。
进一步地，测序产物鉴定位点对比信息如下表所示：
SNP REF ALT
pig18-52722267 T C
pig8-146130825 G T
pig9-10041850 G A
pig13-213464983 C T
表中REF代表参考基因型，ALT代表突变基因型。
进一步地，所述的鉴定位点信息为：各个检测位点的突变基因型与参考基因型，当待测猪检测位点出现参考基因型时认为该位点没有鉴定意义，当待测猪检测位点出现突变基因型时，认为该位点具有鉴定意义。
本发明的有益效果是：与现有技术相比，本发明以浦东白猪品种特有SNP位点为鉴定依据，从分子层面研究鉴别浦东白猪的方法，并以Sanger测序为主要分子鉴定方法，能够将浦东白猪与与常见西方猪种、太湖流域地方品种猪鉴定区分，例如：小梅山猪、枫泾猪、中梅山猪、二花脸猪、嘉兴黑猪、米猪、沙乌头猪、长白猪、大白猪、杜洛克、皮特兰、巴克夏等。
具体实施方式
以下对本发明具体实施方式作进一步详细说明。
本发明涉及一种浦东白猪及生肉制品的SNP标记，包括：pig18-52722267、pig8-146130825、pig9-10041850和pig13-213464983，所述pig18-52722267表示猪18号染色体第52722267位位点，pig8-146130825表示猪8号染色体第146130825位位点，pig9-10041850表示猪9号染色体第10041850位位点，pig13-213464983表示猪13号染色体第213464983位位点。
本发明提供了一种基于上述SNP标记组合的浦东白猪及生肉制品鉴定方法，首先提取待鉴定的生猪肉或肉制品的基因组DNA，然后经PCR扩增后进行琼脂糖凝胶电泳和Sanger测序，得到SNP标记组合各个检测位点的信息，当SNP标记组合任意三个位点处出现特异突变，即可鉴定为浦东白猪及其肉制品。
所述的PCR扩增，其中涉及的引物如表1所示：
表1 扩增位点引物及产物信息
Figure PCTCN2019130973-appb-000001
表中F代表上游引物，R代表下游引物，length代表标准产物长度，F′-position代表SNP位点在扩增产物的位置
所述的PCR扩增，反应体系为模板DNA 1ng/uL、引物1uL、H 2O 3.8uL、2×Taq PCR Masrer Mix 50uL；和/或，所述PCR反应的反应程序为95℃预变性2min，95℃预变性30s，60℃退火温度30s，72℃延伸1min，循环次数30次，72℃再延伸10min。
所述的琼脂糖凝胶的质量浓度为2％。
所述的凝胶电泳，其条带长度要求为如表1所示。
测序产物鉴定位点对比信息如表2所示：
表2 测序产物鉴定位点对比信息
SNP REF ALT
pig18-52722267 T C
pig8-146130825 G T
pig9-10041850 G A
pig13-213464983 C T
表中REF代表参考基因型，ALT代表突变基因型。
如表2所示，各个检测位点的突变基因型与参考基因型，当待测猪检测位点出现参考基因型时认为该位点没有鉴定意义，当待测猪检测位点出现突变基因型时，认为该位点具有鉴定意义，例如一头待测猪A在pig18-52722267位点，如果测序数据为T，则认为待测猪A在pig18-52722267位点不具备鉴定意义；如果测序数据为C，则认为待测猪A在pig18-52722267 位点具有鉴定意义。
由于单个位点作为鉴定依据的存在假阳性错判，且错判概率较高，所以本发明利用位点组合作为鉴定Marker。
各个品种鉴定位点组合鉴定Marker信息如表3所示。
表3 鉴定标记组合信息
Figure PCTCN2019130973-appb-000002
如表3所示，Marker1-4为浦东白猪鉴定标记组合，例如当待测猪A具备4个Marker组合中的任意一种位点突变组合的时候，则认为待测猪A为浦东白猪。
所述的浦东白猪肉制品是指浦东白猪分割肉及以浦东白猪为原料加工制备的腌制品和熟食制品。
随机选取待测猪种耳组织样本5份，采用SDS法提取组织DNA。
利用引物对DNA样品进行PCR反应扩增。
PCR反应的反应体系为10uL体系：模板DNA 1ng/uL、引物1uL、H 2O 3.8uL、2×Taq PCR Masrer Mix 50uL；和/或，PCR反应的反应程序为95℃预变性2min，95℃预变性30s，60℃退火温度30s，72℃延伸1min，循环次数30次，72℃再延伸10min。
用2％的琼脂糖凝胶，1×TAE缓冲液为介质电泳检测扩增结果，凝胶电泳条件为电流10A，电压100伏，时间40分钟。不同引物对扩增产物与表1中的标准产物长度进行比对，产物长度在误差范围内且与标准产物长度一致，则认为扩增结果合格。
将合格的样本PCR扩增产物进行Sanger测序，得到各个检测位点的信息，对测序结果进行分析：
表4 样本测序结果SNP多态性分析表
样品/SNP REF ALT 1 2 3 4 5
pig18-52722267 T C √ √   √  
pig8-146130825 G T   √ √   √
pig9-10041850 G A   √ √   √
pig13-213464983 C T       √ √
表中REF代表参考基因型，ALT代表突变基因型，√代表检测到突变基因型。
如表4所示，由测序数据可得，若只采用单一位点作为鉴定依据，一头待测猪同时归属于多个品种。
利用位点组合Marker的方式来进行鉴定：1号猪在pig18-52722267位点检测到突变，不符合Marker信息，鉴定1号猪不是浦东白猪；2号猪在pig18-52722267、pig8-146130825、pig9-10041850位点检测到突变，符合Marker1信息，鉴定2号猪是浦东白猪；3号猪在pig8-146130825、pig9-10041850位点检测到突变，不符合Marker信息，鉴定3号猪不是浦东白猪；4号猪在pig18-52722267、pig13-213464983位点检测到突变，不符合Marker信息，鉴定4号猪不是浦东白猪；5号猪在pig8-146130825、pig9-10041850、pig13-213464983位点检测到突变，符合Marker4，鉴定5号猪为浦东白猪。
目前国内外没有关于浦东白猪种及其肉制品品种(品系)鉴定的相关专利，将第三代分子标记运用于浦东白猪及其肉制品品种(品系)鉴定，填补了市场的空缺，有效解决了浦东白猪(品系)的鉴伪问题。该鉴定方法相对于现有利用第一代分子标记(RFLP)和第二代分子标记(SSR)做猪种鉴定的专利，具有操作更加简单、结果更加准确、快速高效的优点。同时本发明利用第三代分子标记SNP克服了前两代分子标记可使用位点少的缺点，相对前两代分子标记的鉴别技术来说，也简化了鉴别方法。
上述具体实施可由本领域技术人员在不背离本发明原理和宗旨的前提下以不同的方式对其进行局部调整，本发明的保护范围以权利要求书为准且不由上述具体实施所限，在其范围内的各个实现方案均受本发明之约束。

Claims (5)

  1. 一种浦东白猪及生肉制品的SNP标记组合，其特征在于，包括：pig18-52722267、pig8-146130825、pig9-10041850和pig13-213464983，所述pig18-52722267表示猪18号染色体第52722267位位点，pig8-146130825表示猪8号染色体第146130825位位点，pig9-10041850表示猪9号染色体第10041850位位点，pig13-213464983表示猪13号染色体第213464983位位点。
  2. 一种基于权利要求1所述SNP标记组合的浦东白猪及其生肉制品鉴定方法，其特征在于，首先提取待鉴定的生猪肉或肉制品的基因组DNA，然后经PCR扩增后进行琼脂糖凝胶电泳和Sanger测序，得到SNP标记组合各个检测位点的信息，当SNP标记组合任意三个位点处出现特异突变，即可鉴定为浦东白猪及其肉制品。
  3. 根据权利要求2所述的方法，其特征在于，所述的PCR扩增，pig18-52722267处的上下游引物的序列如SEQ ID NO.1～2所示，pig8-146130825处的上下游引物的序列如SEQ ID NO.3～4所示，pig9-10041850处的上下游引物的序列如SEQ ID NO.5～6所示，pig13-213464983处的上下游引物的序列如SEQ ID NO.7～8所示。
  4. 根据权利要求2所述的方法，其特征在于，测序产物鉴定位点对比信息如下表所示：
    SNP REF ALT pig18-52722267 T C pig8-146130825 G T pig9-10041850 G A pig13-213464983 C T
    表中REF代表参考基因型，ALT代表突变基因型。
  5. 根据权利要求4所述的方法，其特征在于，所述的鉴定位点信息为：各个检测位点的突变基因型与参考基因型，当待测猪检测位点出现参考基因型时认为该位点没有鉴定意义，当待测猪检测位点出现突变基因型时，认为该位点具有鉴定意义。
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