WO2019218743A1 - 一种近交系遗传质量监控的snp快速检测方法和snp位点及其引物 - Google Patents

一种近交系遗传质量监控的snp快速检测方法和snp位点及其引物 Download PDF

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WO2019218743A1
WO2019218743A1 PCT/CN2019/075790 CN2019075790W WO2019218743A1 WO 2019218743 A1 WO2019218743 A1 WO 2019218743A1 CN 2019075790 W CN2019075790 W CN 2019075790W WO 2019218743 A1 WO2019218743 A1 WO 2019218743A1
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snp
primer
c57bl
inbred
quality monitoring
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赵静
琚存祥
马秀英
张明坤
侯欢欢
高翔
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江苏集萃药康生物科技有限公司
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  • the invention belongs to the field of genetic background identification and genetic pollution detection of inbred mouse strains, and relates to a rapid detection method of SNP for genetic quality monitoring of inbred lines, in particular to a method for detecting SNP typing based on KASP method and SNP position. Point and its primers.
  • the quality control of experimental animals is the core issue in the healthy development of experimental animal industry. Microbial quality control and genetic background quality control are important control factors. Domestic genetic quality monitoring lacks mature industry standards, and it is essential to establish a fast and accurate high-throughput genotyping technology platform.
  • biochemical marker analysis There are three main methods for quality control detection in genetic background: biochemical marker analysis, microsatellite DNA, and SNP (single nucleotide polymorphism) detection.
  • the international genetic testing method is biochemical labeling method. This method is to detect changes in isozymes or isomeric proteases to infer the corresponding gene changes. Using this method for detection, there is low accuracy, low sensitivity, and detection. The disadvantages are limited, reflecting the limited genetic profile. Molecular genetic markers can be used for more precise regulation of experimental animals. It is a more complete method for detecting animal quality.
  • SNP detection is a technique of molecular genetic markers to detect single nucleotide variation at the genome level. The resulting DNA sequence polymorphism; SNPs can detect single-base variation, high density, representativeness, genetic stability, etc., and can fully reflect the genetic and variation of the genome.
  • the KASP method refers to the Kompetitive Allele Specific PCR, which performs precise biallelic genotyping of target SNPs based on high-sensitivity fluorescence detection. Unlike traditional Taqman technology, this method does not require labeling of specific targets, ie, specific primers/probes, and does not require the synthesis of specific fluorescent primers for each SNP site. Its unique ARM PCR principle allows all positions. Point detection is ultimately amplified using universal fluorescent primers, greatly reducing the cost of the experiment. The optimized PCR system can meet the requirements of high-throughput reactions at different sites. It has the accuracy of the gold standard and reduces the cost of use. It has better site adaptability than Taqman. KASP technology combines two reactions of traditional detection alleles into one reaction with low cost. SNPs detection not only makes up for the shortcomings of traditional PCR, gel cutting and sequencing processes, but also saves the cost of sequencing.
  • the current SNP detection panel is limited by the operation technique and the detection cost, resulting in a low sample detection flux and limited selection of sites.
  • the present invention aims to provide a high throughput. , multi-site, low-cost, rapid SNP rapid detection method for inbred genetic quality monitoring
  • a rapid SNP detection method for genetic quality monitoring of inbred lines includes the following steps:
  • Design of SNP panel Determine the inbred mouse strain, and screen out the specific genetic quality monitoring sites of C57BL/6 strain and other strains, and the specific discrimination sites are as follows: The base of the C57BL/6 strain site is different from the corresponding base in the 129S1/SvIm, BALB/C, A/J, CBA, DBA, FVB, NOD strains. In the chromosome unit, the SNP panel should include at least 5 pairs.
  • Chromosomes containing specific discrimination sites, and each pair of chromosomes should contain more than 2 specific discrimination sites;
  • the conventional genetic quality monitoring sites of the strains are as follows: on the basis of the specific discrimination sites, according to each pair The principle of 4-5 loci on the chromosome, complementing the loci at equal intervals, complementing the SNP panel locus of the C57BL/6 strain to 96; the SNP panel can be used for C57BL strain inbred mice and corresponding mutations Detection of genetic quality and strain contamination in mice and effective identification of C57BL strain mice and other strains;
  • the ends of the primer precursor are respectively two mutated bases of the SNP panel site; the sequence recognizing the FAM or HEX signal is set at the 5' end of the primer precursor, and the length is about 20 bp; the 3' end downstream primer One, which is about 18-29 bp in length;
  • the sample template contained the base mutation type; the 96 lines were used for the control line 129S1/Svlm subline 129S1/SvlmJNju and C57BL/ The 6 subfamily C57BL/6JNju was tested for primers. If the typing was successful, the primers were tested successfully. Otherwise, the sites where the test failed were located upstream and downstream of the corresponding positions on the chromosome; the 96 sites and PCR primers were successfully tested. The test conditions and system are determined as the final test plan;
  • the inbred mouse strains are A/J, 129S1/SvIm, BALB/C, C57BL/6, CBA, DBA, FVB, NOD; the C57BL/6 strain and other
  • the specificity of the seven lines has 69 distinguishable loci, as follows:
  • the concentration requirement of the sample DNA to be tested is 5 ng/uL ⁇ the genome size of the test species/human genome size.
  • reaction system of the KASP method is as follows: including DNA (5-50 ng/uL, taking human or mouse as an example) in a volume of 0.8 uL per reaction well, 2 ⁇ KASP Master mix in each reaction well The volume is 0.778 uL and the volume of the KASP Primer mix in each well is 0.022 uL.
  • reaction procedure of the KASP method is as follows:
  • a set of 96 loci were used as SNP loci in inbred genetic quality surveillance, the 96 loci being as follows:
  • a SNP site primer for inbred genetic quality monitoring the primer sequences are set forth in SEQ ID NO. 1 to SEQ ID NO.
  • the detection method of the present invention completes the site detection of 36 samples of one project (96 sites per sample), and the total period required is only 6 working days.
  • the method of the invention improves the number of detectable sites of a single sample, and the high-throughput detection greatly shortens the experimental period, and further improves the throughput while ensuring the accuracy of the results.
  • the C57BL SNP panel used in the present invention contains 96 sites, and can be used for A/J, 129S1/SvIm, BALB/C, CBA, DBA, FVB, except for routine genetic quality monitoring of C57BL strains and related mutant lines. , the distinction of NOD strains.
  • the invention does not need to label the target, ie, the specific primer, does not need to design the probe, and does not need to synthesize a specific fluorescent primer according to each SNP site, and all the site detections are finally amplified by the universal fluorescent primer, and it is not required.
  • the traditional PCR process of running glue, cutting, sequencing, etc. saves the detection time and greatly saves the cost of sequencing; in addition, the KASP method performs biallelic genotyping of target SNPs, and two reactions of traditional detection alleles One reaction was synthesized and the cost was reduced.
  • Figure 1 is a flow chart of the detection method of the present invention.
  • the SNP rapid detection method for the inbred genetic quality monitoring of the present embodiment is shown in Figure 1.
  • the specific steps are as follows:
  • the C57BL/6 strain SNP panel can be used for routine SNP detection for differentiation from A/J, 129S1/SvIm, BALB/C, CBA, DBA, FVB, NOD strains; it can also be used for C57BL/6N, Routine genetic quality monitoring of C57BL lines and related mutant lines such as C57BL/10, C57BLKS, B6(Cg)-Tyr c-2J .
  • SNP panel design is mainly carried out in two aspects: screening sites that can be specifically distinguished from other strains - for strain contamination detection; screening strains for routine genetic quality monitoring sites.
  • the SNP panel should include at least 5 pairs of chromosomes with specific discrimination sites, and each pair of chromosomes should contain more than 2 specific discrimination sites, then the specific distinction between panels can be considered Line mice are distinguished from other inbred lines. Sites can be specifically distinguished: in a line, the site is not the same as other strain sites. If a SNP detection site cannot be completely distinguished from other strains, if a mutation occurs after the test, it is impossible to determine whether it is genetic drift or strain contamination.
  • the frequency of genetic quality monitoring is 1 year/time. If pollution occurs, the longest pollution period should be 1 year, and the contaminated mouse algebra can be up to 4 generations.
  • the probability of chromosomal contamination in the fourth generation of mice was at least 5 (according to the F1 generation and other strains, the subsequent mice were all backcrossed with pure background lines.
  • the F2 generation contaminated chromosomes were all, and the F3 contaminated chromosomes were 10 Article, the number of chromosomes contaminated by F4 is 5).
  • the panel can distinguish the strain mice from other inbred lines.
  • the detection results are informative when there are more than 2 sites on each chromosome.
  • Line-specific discrimination sites were screened using a programmatic approach, and the screening principle was in the same line, screening sites that were not identical to other lines.
  • the selected sites are artificially screened according to the principle of equidistant distance on the chromosome (the number of sites on each chromosome is 4-5, the number of specific sites should be no less than 2), and the specific differentiation of each line is determined. Panel.
  • the regular sites are complemented according to the principle of 4-5 equally spaced loci on each chromosome; at the same time, the sites with multiple applicability in multiple varieties of panels are preferentially selected.
  • the 100 bp sequences of the upstream and downstream sequences of the SNP locus are extracted in the mouse genome sequence using a programming tool; the 5' primer design: 20-30 bp primers are designed upstream of the SNP locus.
  • the ends of the primers are two mutated bases of the SNP, and the 5' end of the primer is added with a sequence of about 20 bp to recognize different signals, such as FAM and HEX signals; and the 3' design downstream primer is about 18-29 bp in length.
  • Tm (°C) is between 55-65 ° C
  • GC% is between 34%-60%
  • a total of 288 primers are designed.
  • the specific primer sequences are shown in the table below. A 96-site PCR amplification assay was performed for each sample, and 3 primers at each site were simultaneously subjected to PCR amplification. If one of the signals was generated in the system, the sample template contained the base mutation type.
  • the tail-tail DNA of inbred mice was extracted using LGC's oKtopureTM high-throughput DNA extractor.
  • the extraction procedure refers to the instrument description. Can effectively increase the DNA extraction flux, 3,500 samples / day; extraction speed: 20-30mg starting sample size, 8 ⁇ 96 ⁇ 1.5h, 80-100mg starting sample size, 8 ⁇ 96 ⁇ 2h. 2, DNA concentration requirements
  • the reaction system was automatically prepared using LGC's IntelliQube instrument.
  • the KASP genotyping reaction system is shown in the following table:
  • the PCR reaction was carried out by the KASP method using a Hydrocycler 2 TM water bath PCR thermal cycle water bath system of LGC.
  • the KASP genotyping reaction program can be divided into the following three types, and any one can complete the operation, as shown in Table 6-8.
  • Readings were performed using IntelliQube fluorescence detection, and data analysis after PCR was performed using IntelliScore, and genotypes were automatically derived for analysis.
  • the detection steps and methods are the same, except that the sample DNA is different.
  • a total of 10 strains were selected, including C57BL-related strains and C57BL/6 mutant mice, for SNP locus detection.
  • the genetic quality of the mice was examined.
  • the results showed that C57BL/10JNju mice had a locus on chromosome 18, rs3725940 and NCBI.
  • the database was inconsistent and homozygous for mutation; in the C57BL/6 mutant mice, the sites of 9 lines were consistent with C57BL/6J, as shown in the table below.

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Abstract

提供一种近交系遗传质量监控的SNP快速检测方法，首先设计SNP panel，然后设计SNP位点引物，再提取样本DNA，用KASP法进行基因分型，并与NCBI上的SNP位点信息比较，完成近交系小鼠遗传质量监控。还提供一种近交系遗传质量监控的SNP位点及SNP位点引物。

Description

一种近交系遗传质量监控的SNP快速检测方法和SNP位点及其引物 技术领域
本发明属于近交系小鼠品系遗传背景鉴定与遗传污染检测领域，涉及一种近交系遗传质量监控的SNP快速检测方法，尤其涉及一种基于KASP法进行SNP分型的检测方法及SNP位点和其引物。
背景技术
实验动物质量控制是实验动物行业健康发展的核心问题，小鼠微生物质量控制及遗传背景质量控制是其中的重要控制因素。国内的遗传质量监控缺少成熟的行业标准，建立一种快速且精准性高的高通量基因分型技术平台至关重要。
用于遗传背景质量控制检测方法主要有3种：生化标记分析法、微卫星DNA、SNP(单核苷酸的多态性)检测等。目前国际所规定的遗传检测方法是生化标记分析法，这一方法是检测同工酶或异构蛋白酶的变化来推测相应的基因变化；使用此方法进行检测，存在准确度低、灵敏度低、检测位点有限、反映遗传概貌有限等弊端。而分子遗传标记可以对实验动物进行更精细的监管，是一种更完善的实验动物质量检测手段；其中SNP检测作为分子遗传标记的一门技术，在基因组水平上检测由单个核苷酸的变异所引起的DNA序列多态性；SNP所表现的多态性可以监测到单个碱基的变异，具有密度高、代表性、遗传稳定性等特点，能够全面地反映基因组的遗传及变异情况。
KASP法是指竞争性等位基因特异性PCR(Kompetitive Allele Specific PCR)，以高灵敏度的荧光检测为基础，对目标SNPs进行精准的双等位基因分型。与传统Taqman技术不同的是，此种方法无需对靶点即特异性引物/探针进行标记，不需要根据每个SNP位点合成特异的荧光引物，其独特的ARM PCR原理，让所有的位点检测最终都使用通用荧光引物扩增，大大降低了实验成本。优化的PCR体系可满足不同位点高通量反应的需求，既有金标准的准确，又降低了使用成本，比Taqman具有更好的位点适应性。KASP技术将传统检测等位基因的2个反应合成了1个反应，成本低。SNPs检测不但弥补了传统PCR、切胶、测序流程时间长的缺点，而且大大节省了测序的费用。
发明内容
目前的SNP检测panel由于受到操作技术及检测成本的制约，导致样本检测通量较低、位点的选择受到限制，针对现有技术中存在的缺点，本发明的目的在于提供一种高通量、多位点、低成本、快速用于近交系遗传质量监控的SNP快速检测方法
本发明的目的还在于提供一组位点在近交系遗传质量监控中作为的SNP位点的应用。
本发明的目的还在于提供一种近交系遗传质量监控的SNP位点引物。
本发明解决其技术问题所采用的技术方案是：
一种近交系遗传质量监控的SNP快速检测方法，包括如下步骤：
(1)SNP panel的设计：确定近交系小鼠品系，筛选出C57BL/6品系与其它品系的可特异性区分位点和品系常规遗传质量监控位点，所述特异性区分位点如下：C57BL/6品系位点的碱基与129S1/SvIm、BALB/C、A/J、CBA、DBA、FVB、NOD品系中对应的碱基不同，以染色体为单位，SNP panel中应包括至少5对含特异性区分位点的染色体，且每对染色体应含有2个以上特异性区分位点；所述品系常规遗传质量监控位点如下：在所述特异性区分位点的基础上，按照每对染色体上4-5个位点原则，等间距地进行位点的补足，将C57BL/6品系的SNP panel位点补足为96个；所述SNP panel可用于C57BL品系近交系小鼠及相应突变系小鼠的遗传质量检测及品系污染情况检测以及C57BL品系小鼠与其他品系的有效鉴别；
(2)设计并合成上述SNP panel位点的引物，不需设计探针；SNP panel位点上下游序列各100bp序列使用编程工具在小鼠基因组序列中进行拉取；其中，5’端上游引物设计：5’端上游引物共有两条，均包括引物前体和一段可识别FAM或HEX信号的序列；所述引物前体设在每个SNP panel位点的上游，长度为20-30bp，在引物前体的末端分别为SNP panel位点的两个突变碱基；所述可识别FAM或HEX信号的序列设在所述引物前体的5’端，长度约为20bp；3’端下游引物为一条，其长度约18-29bp；
将上述3条引物同时进行PCR扩增，若体系中产生其中一个信号，则说明样品模板中含有该碱基突变类型；对96个位点使用对照品系129S1/Svlm亚系129S1/SvlmJNju及C57BL/6亚系C57BL/6JNju进行引物测试，若成功分型，则表示引物测试成功，否则对测试失败的位点在染色体相应位置的上下游寻找替换 位点；测试成功的96个位点及PCR引物测试条件及体系确定为最终检测方案；
(3)提取待测样本DNA，样品浓度大于10ng/uL时，用KASP法对每个样本的96个SNP panel位点进行基因分型，分析数据并与NCBI上的SNP位点信息比较，完成近交系小鼠遗传质量监控与品系鉴定。
进一步地，所述步骤(1)中，近交系小鼠品系为A/J、129S1/SvIm、BALB/C、C57BL/6、CBA、DBA、FVB、NOD；所述C57BL/6品系与其他7个品系的特异性可区分位点有69个，具体如下：
RS_ID 129S1/SvImJ A/J BALB/cJ C57BL/6J CBA/CaJ DBA/1J FVB/NJ NOD/LtJ
rs3725641 C C C T C C C C
rs3022803 C C C A C C C C
rs4136901 C C C A C C C C
rs3720900 G G G A G G G G
rs3686727 G G G A G G G G
rs3726475 A A A T A A A A
rs3024096 G G G C G G G G
rs3659459 T T T A T T T T
rs3022965 G G G A G G G G
rs3725806 T T T A T T T T
rs4138316 T T T C T T T T
rs3654495 T T T C T T T T
rs3664065 A A A C A A A A
rs3680364 T T T G T T T T
rs3703682 A A A C A A A A
rs4225380 C C C T C C C C
rs3670250 A A A G A A A A
rs4225536 G G G A G G G G
rs3023064 T T T G T T T T
rs3664216 T T T C T T T T
rs3723352 T T T G T T T T
rs3684061 A A A C A A A A
rs3720603 A A A G A A A A
rs3656205 G G G A G G G G
rs3705245 C C C T C C C C
rs3716088 G G G T G G G G
rs3664394 C C C A C C C C
rs3664354 A A A G A A A A
rs3683511 G G G A G G G G
rs3674352 T T T C T T T T
rs3716314 C C C A C C C C
rs3706877 G G G A G G G G
rs3673457 T T T C T T T T
rs3657415 T T T C T T T T
rs3663844 A A A G A A A A
rs3696310 T T T C T T T T
rs3696307 C C C G C C C C
rs3723733 A A A G A A A A
rs3686921 A A A C A A A A
rs3090226 T T T C T T T T
rs3675087 G G G A G G G G
rs3023949 G G G A G G G G
rs3091105 C C C T C C C C
rs3706319 T T T C T T T T
rs3701757 G G G A G G G G
rs3724682 G G G A G G G G
rs3023384 T T T C T T T T
rs3666540 A A A G A A A A
rs3724755 G G G A G G G G
rs3696080 G G G T G G G G
rs3697794 A A A G A A A A
rs3689061 A A A G A A A A
rs3715673 T T T C T T T T
rs3702158 G G G A G G G G
rs3023422 G G G A G G G G
rs4140226 A A A G A A A A
rs3090912 T T T G T T T T
rs3660203 G G G C G G G G
rs3684506 G G G A G G G G
rs3654800 G G G A G G G G
rs3706369 T T T C T T T T
rs3725940 T T T G T T T T
rs3726735 G G G A G G G G
rs3679049 A A A G A A A A
rs3725703 C C C G C C C C
rs3699591 C C C T C C C C
rs3157180 C C C T C C C C
rs3690903 G G G T G G G G
rs3715531 G G G A G G G G
。
进一步地，所述步骤(3)中，待测样本DNA的浓度需求＝5ng/uL×待测物种基因组大小/人类基因组大小。
进一步地，所述KASP法的反应体系如下：包括DNA(5-50ng/uL，以人或小鼠为例)在每个反应孔的体积为0.8uL，2×KASP Master mix在每个反应孔的体积为0.778uL，KASP Primer mix在每个反应孔的体积为0.022uL。
进一步地，所述KASP法的反应程序如下：
Figure PCTCN2019075790-appb-000001
或
Figure PCTCN2019075790-appb-000002
或
Figure PCTCN2019075790-appb-000003
一组96个位点在近交系遗传质量监控中作为SNP位点的应用，所述96个位点如下：
Figure PCTCN2019075790-appb-000004
Figure PCTCN2019075790-appb-000005
Figure PCTCN2019075790-appb-000006
*为特异性区分位点。
一种近交系遗传质量监控的SNP位点引物，所述引物序列如SEQ ID NO.1至SEQ ID NO.288所示。
本发明具有的有益效果如下：
本发明检测方法完成1个项目的36个样品的位点检测(每个样品检测96个位点)，需要的总周期仅仅为6个工作日。本发明方法提高了单个样品的可检测位点数、而且高通量检测使得实验周期大大缩短，此外在提高通量的同时保证了结果的准确性。
本发明使用的C57BL SNP panel共含有96个位点，除用于C57BL品系及相关突变品系的常规遗传质量监控外，可用于与A/J、129S1/SvIm、BALB/C、CBA、DBA、FVB、NOD品系的区分。
本发明使用无需对靶点即特异性引物进行标记，不需要设计探针，不需要根据每个SNP位点合成特异的荧光引物，所有的位点检测最终都使用通用荧光引物扩增，不需要传统PCR的跑胶、切胶、测序等流程，节省了检测时间，而且大大节省了测序的费用；此外KASP法对目标SNPs进行双等位基因分型，将传统检测等位基因的2个反应合成了1个反应，成本降低。
附图说明
图1为本发明检测方法的流程图。
具体实施方式
下面结合附图和实施例对本发明做进一步的说明。
本实施例的近交系遗传质量监控的SNP快速检测方法，其流程如图1所示，具体步骤详见如下：
实施例1：SNP panel设计
1、确定SNP panel中包含的近交品系
选取13种近交系小鼠品系进行常规SNP检测(如表1)，13种近交系小鼠品系均由南京大学-南京生物医药研究院(NBRI)提供(公开销售)，在筛选过程中发现，C57BL/6J与C57BL/6N、C57BL/10J、C57BLKS/J、B6(Cg)-Tyr c-2J遗传背景相似；同时，DBA/1与DBA/2遗传背景相似。因此，C57BL中选取C57BL/6J作为代表，DBA中选取DBA/1作为代表，最终确定8个近交系来建立C57BL/6近交品系的SNP检测panel(如表2)。在后续实验中C57BL/6品系SNP panel可用于常规SNP检测，用于与A/J、129S1/SvIm、BALB/C、CBA、DBA、FVB、NOD品系的区分；同时可以用于C57BL/6N、C57BL/10、C57BLKS、B6(Cg)-Tyr c-2J等C57BL品系及相关突变品系的常规遗传质量监控。
表1 研究院近交系列表
序号 品系名称
1 C57BL/6NNju
2 C57BL/6JNju
3 CBA/CaJNju
4 DBA/2JNju
5 A/JNju
6 BALB/cJNju
7 C57BL/10JNju
8 FVB/NJNju
9 C57BLKS/JNju
10 DBA/1JNju
11 NOD/ShiLtJNju
12 B6(Cg)-Tyr c-2J/Nju
13 129S1/SvImJNju
表2 用于建立C57BL/6SNP检测panel的近交系列表
序号 品系名称
1 CBA/CaJNju
2 C57BL/6JNju
3 A/Jnju
4 BALB/cJNju
5 FVB/NJNju
6 DBA/1JNju
7 NOD/ShiLtJNju
8 129S1/SvImJNju
2、SNP Panel设计方向
以LGC公司提供的数据库<mouse-marker-excel-file>进行筛选。SNP panel设计主要从两大方面进行：筛选出与其它品系能够特异性区分的位点——用于品系污染检测；筛选品系常规遗传质量监控的位点。
3、筛选近交系与其它品系间特异性区分的位点
a)筛选目的：用于品系污染检测。
b)设计原则：以染色体为单位，SNP panel中应包括至少5对含特异性区分位点的染色体，且每对染色体应含有2个以上特异性区分位点，则认为特异性区分panel可将品系小鼠与其它近交系进行区分。可特异性区分位点：在某一品系中，位点与其它品系位点均不相同。如果一个SNP检测位点不能够完全与其它品系进行区分，检测后若出现突变，则不能确定是遗传漂变还是品系污染。
遗传质量监控的频率为1年/次，若出现污染，则最长污染周期应为1年，污染小鼠代数最长为4代。第四代小鼠出现染色体污染的概率最低为5条(按照F1代与其它品系配繁，后续小鼠全部与纯背景品系进行回交计算。则F2代污染染色体为全部，F3污染染色体为10条，F4污染染色体条数为5条)。
以此概率进行计算，若5条以上染色体含有特异性位点，则panel能够将品系小鼠与其它近交系进行区分。每条染色体上含有2个以上位点则认为检测结果具有参考性。
c)筛选结果：C57BL/6近交系小鼠共筛选出69个与其他品系可特异性区分位点。该panel用于C57BL品系及相关突变系的SNP检测，可用于与A/J、129S1/SvIm、BALB/C、CBA、DBA、FVB、NOD品系的区分。
品系特异性区分位点使用编程的方式进行筛选，筛选原则为同一品系中，筛选与其它品系均不相同的位点。筛选出的位点根据在染色体上等间距的原则进行人工二次筛选(每条染色体上位点数为4-5个，特异性位点数应不少于2个)，确定每个品系的特异性区分panel。
4、筛选品系常规遗传质量监控的位点
a)筛选目的：用于品系常规遗传质量监控。
b)设计原则：品系特异性位点通常数量过少，不够用于常规遗传质量监控。在确定上述特异性区分位点panel的基础上，我们将每个品系检测panel SNP数量补足为96个，其作用如下：既可以保证SNP检测结果可靠性，又方便鉴定操作。
在特异性位点的基础上，按照每条染色体上4-5个等间距位点原则进行常规位点的补足；同时，优先选取在多种品系panel中多适用性的位点。
c)筛选结果：共筛选出包含96个位点的C57BL/6J品系遗传质量监控SNP检测panel，如下表(即表3)所示。
表3 C57BL/6品系遗传质量监控SNP检测panel
Figure PCTCN2019075790-appb-000007
Figure PCTCN2019075790-appb-000008
Figure PCTCN2019075790-appb-000009
备注：*为特异性区分位点。
实施例2：SNP位点引物设计
SNP位点筛选完成后，SNP位点上下游序列各100bp序列使用编程工具在小鼠基因组序列中进行拉取；5’端引物设计：在SNP位点的上游分别设计20-30bp的引物，在引物的末端分别为SNP的两个突变碱基，引物5’端增加一段约20bp的分别识别不同信号的序列，如FAM及HEX信号；3’设计下游引物，长度约18-29bp。
Tm(℃)在55-65℃之间，GC％在34％-60％之间，共设计引物288条，具体引物序列详见下表。对每个样品安排96个位点的PCR扩增检测，每个位点的 3条引物同时进行PCR扩增，若体系中产生其中一个信号，则说明样品模板中含有该碱基突变类型。
表4 位点引物信息
Figure PCTCN2019075790-appb-000010
Figure PCTCN2019075790-appb-000011
Figure PCTCN2019075790-appb-000012
Figure PCTCN2019075790-appb-000013
Figure PCTCN2019075790-appb-000014
Figure PCTCN2019075790-appb-000015
Figure PCTCN2019075790-appb-000016
Figure PCTCN2019075790-appb-000017
Figure PCTCN2019075790-appb-000018
实施例3：KASP法进行基因分型
1、提取待检测样品的DNA作为模板
使用LGC公司的oKtopureTM高通量DNA提取仪提取近交系小鼠的鼠尾DNA。提取步骤参照仪器说明。可有效提高DNA的提取通量，3,500个样本/天；提取速度：20-30mg起始样本量，8×96≤1.5h，80-100mg起始样本量，8×96≤2h。2、DNA浓度需求
大多数KASP检测的每个反应需要5-50ng DNA；DNA浓度需求依据基因组大小变化：待测物种DNA浓度需求＝5ng/uL×待测物种基因组大小/人类基因组大小。
3、PCR反应体系的配制
使用LGC公司的IntelliQube仪器自动进行反应体系的配制。KASP基因分型反应体系，如下表所示：
表5 KASP基因分型反应体系
Figure PCTCN2019075790-appb-000019
4、PCR反应
PCR反应选用KASP法，使用LGC公司的Hydrocycler2 TM水浴PCR热循环仪水浴系统进行PCR反应。KASP基因分型反应程序可分为以下三种，任意一种均可完成操作，如表6-8所示。
表6 KASP基因分型反应程序(1)
Figure PCTCN2019075790-appb-000020
Figure PCTCN2019075790-appb-000021
或 表7 KASP基因分型反应程序(2)
Figure PCTCN2019075790-appb-000022
或 表8 KASP基因分型反应程序(3)
Figure PCTCN2019075790-appb-000023
5、高通量基因分型及数据分析
使用IntelliQube荧光检测进行读数，使用IntelliScore进行PCR后的数据分析，自动导出基因型进行分析。
6、对C57BL/6亚系C57BL/6JNju小鼠进行SNP分型鉴定
以129S1/SvImJNju小鼠作为对照品系，对C57BL/6JNju进行96个位点的引物测试及小鼠的基因分型；结果表明C57BL/6JNju在96个位点中与NCBI上登记的位点信息全部一致，详见下表9。
表9 C57BL/6JNju亚系SNP检测结果
RS号 染色体 C57BL/6JNju 129S1/SvImJNju NCBI C57BL/6J
rs3664394 8 A C A
rs3706319 12 C T C
rs3706076 12 C C C
rs3697794 14 G A G
rs3022803 1 A C A
rs3664528 1 C T C
rs4136901 1 A C A
rs3701379 2 G A G
rs3687947 2 C G C
rs3704086 3 C C C
rs4138316 4 C T C
rs3654495 4 C T C
rs16265 4 G A G
rs3664065 4 C A C
rs3680364 4 G T G
rs3659100 5 C C C
rs4226520 7 C T C
rs3720603 7 G A G
rs3705245 7 T C T
rs3716088 7 T G T
rs3694533 9 T C T
rs3716314 9 A C A
rs3706877 9 A G A
rs3657415 9 C T C
rs3663844 10 C T C
rs3705913 10 G A G
rs3693721 10 G T G
rs3723733 11 G A G
rs3090226 11 C T C
rs3675087 11 A G A
rs3023949 12 T C T
rs3091105 12 G G G
rs4229611 12 A G A
rs3023384 13 C T C
rs3666540 13 G A G
rs3724755 13 A G A
rs3667842 14 G T G
rs3656384 15 G A G
rs3090912 16 G T G
rs4167317 16 C G C
rs4183127 16 T C T
rs3721713 17 G A G
rs3706767 18 T C T
rs3654800 18 A G A
rs3706369 18 C T C
rs3692733 19 A A A
rs3679049 19 G A G
rs3653863 X A G A
rs3720900 1 A G A
rs3703682 5 C A C
rs3723352 6 G T G
rs3674352 8 C T C
rs3696310 10 C T C
rs3715673 14 C T C
rs3702158 15 A G A
rs3660203 17 C G C
rs3684506 17 A G A
rs3726735 19 A G A
rs3699591 X T C T
rs3715531 X A G A
rs3684061 6 C A C
rs3683511 8 A G A
rs3696307 10 G C G
rs4140226 15 G A G
rs3023422 15 A G A
rs3725641 1 T C T
rs3726475 2 T A T
rs3024096 2 C G C
rs3680834 3 G C G
rs3659459 3 A T A
rs3022965 3 T C T
rs3725806 3 A T A
rs4225380 5 A G A
rs3664216 6 C T C
rs3725568 6 C T C
rs3656205 7 A G A
rs3664354 8 G A G
rs3711293 8 C C C
rs3673457 9 C T C
rs3023989 11 T T T
rs3696080 14 T G T
rs3689286 15 T G T
rs4205499 16 T C T
rs3703141 17 G C G
rs3725940 18 G T G
rs3725703 19 G C G
rs3157180 X T C T
rs3690903 X A C A
rs3724682 13 A G A
rs3686727 2 A G A
rs3670250 5 G A G
rs4225536 5 A G A
rs3023064 6 G T G
rs3686921 11 C A C
rs3701757 13 A G A
rs3689061 14 G A G
实施案例：
检测步骤及方法均一致，不同的是样本DNA不同。共选取10个品系，分别 包含C57BL相关品系以及C57BL/6的突变系小鼠进行SNP位点检测，查看小鼠遗传质量情况，结果显示C57BL/10JNju小鼠18号染色体上一个位点rs3725940与NCBI数据库不一致，且为突变纯合；C57BL/6突变系小鼠中，9个品系的位点与C57BL/6J一致，具体见下表。
表10 10个C57BL近交系及突变系小鼠SNP检测结果
Figure PCTCN2019075790-appb-000024

Claims (7)

  1. 一种近交系遗传质量监控的SNP快速检测方法，其特征在于，包括如下步骤：
    (1)SNP panel的设计：确定近交系小鼠品系，筛选出C57BL/6品系与其它品系的可特异性区分位点和品系常规遗传质量监控位点，所述特异性区分位点如下：C57BL/6品系位点的碱基与129S1/SvIm、BALB/C、A/J、CBA、DBA、FVB、NOD品系中对应的碱基不同，以染色体为单位，SNP panel中应包括至少5对含特异性区分位点的染色体，且每对染色体应含有2个以上特异性区分位点；所述品系常规遗传质量监控位点如下：在所述特异性区分位点的基础上，按照每对染色体上4-5个位点原则，等间距地进行位点的补足，将C57BL/6品系的SNP panel位点补足为96个；所述SNP panel可用于C57BL品系近交系小鼠及相应突变系小鼠的遗传质量检测及品系污染情况检测以及C57BL品系小鼠与其他品系的有效鉴别；
    (2)设计并合成上述SNP panel位点的引物，不需设计探针；SNP panel位点上下游序列使用编程工具在小鼠基因组序列中进行拉取；其中，5’端上游引物设计：5’端上游引物共有两条，均包括引物前体和一段可识别FAM或HEX信号的序列；所述引物前体设在每个SNP panel位点的上游，长度为20-30bp，在引物前体的末端分别为SNP panel位点的两个突变碱基；所述可识别FAM或HEX信号的序列设在所述引物前体的5’端，长度约为20bp；3’端下游引物为一条，其长度约18-29bp；
    将上述3条引物同时进行PCR扩增，若体系中产生其中一个信号，则说明样品模板中含有该碱基突变类型；对96个位点使用对照品系进行引物测试，若成功分型，则表示引物测试成功，否则对测试失败的位点在染色体相应位置的上下游寻找替换位点；测试成功的96个位点及PCR引物测试条件及体系确定为最终检测方案；
    (3)提取待测样本DNA，样品浓度大于10ng/uL时，用KASP法对每个样本的96个SNP panel位点进行基因分型，分析数据并与NCBI上的SNP位点信息比较，完成近交系小鼠遗传质量监控与品系鉴定。
  2. 如权利要求1所述的一种近交系遗传质量监控的SNP快速检测方法，其特征在于，所述步骤(1)中，近交系小鼠品系为A/J、129S1/SvIm、BALB/C、C57BL/6、CBA、DBA、FVB、NOD；所述C57BL/6的亚系C57BL/6J品系与其他7个品系的特异性可区分位点有69个，具体如下：
    RS_ID 129S1/SvImJ A/J BALB/cJ C57BL/6J CBA/CaJ DBA/1J FVB/NJ NOD/LtJ rs3725641 C C C T C C C C rs3022803 C C C A C C C C rs4136901 C C C A C C C C rs3720900 G G G A G G G G rs3686727 G G G A G G G G rs3726475 A A A T A A A A rs3024096 G G G C G G G G rs3659459 T T T A T T T T rs3022965 G G G A G G G G rs3725806 T T T A T T T T rs4138316 T T T C T T T T rs3654495 T T T C T T T T rs3664065 A A A C A A A A rs3680364 T T T G T T T T rs3703682 A A A C A A A A rs4225380 C C C T C C C C rs3670250 A A A G A A A A rs4225536 G G G A G G G G rs3023064 T T T G T T T T rs3664216 T T T C T T T T rs3723352 T T T G T T T T rs3684061 A A A C A A A A rs3720603 A A A G A A A A rs3656205 G G G A G G G G rs3705245 C C C T C C C C rs3716088 G G G T G G G G rs3664394 C C C A C C C C rs3664354 A A A G A A A A rs3683511 G G G A G G G G rs3674352 T T T C T T T T rs3716314 C C C A C C C C rs3706877 G G G A G G G G rs3673457 T T T C T T T T rs3657415 T T T C T T T T rs3663844 A A A G A A A A rs3696310 T T T C T T T T rs3696307 C C C G C C C C rs3723733 A A A G A A A A rs3686921 A A A C A A A A rs3090226 T T T C T T T T rs3675087 G G G A G G G G
    rs3023949 G G G A G G G G rs3091105 C C C T C C C C rs3706319 T T T C T T T T rs3701757 G G G A G G G G rs3724682 G G G A G G G G rs3023384 T T T C T T T T rs3666540 A A A G A A A A rs3724755 G G G A G G G G rs3696080 G G G T G G G G rs3697794 A A A G A A A A rs3689061 A A A G A A A A rs3715673 T T T C T T T T rs3702158 G G G A G G G G rs3023422 G G G A G G G G rs4140226 A A A G A A A A rs3090912 T T T G T T T T rs3660203 G G G C G G G G rs3684506 G G G A G G G G rs3654800 G G G A G G G G rs3706369 T T T C T T T T rs3725940 T T T G T T T T rs3726735 G G G A G G G G rs3679049 A A A G A A A A rs3725703 C C C G C C C C rs3699591 C C C T C C C C rs3157180 C C C T C C C C rs3690903 G G G T G G G G rs3715531 G G G A G G G G
    。
  3. 如权利要求1或2所述的一种近交系遗传质量监控的SNP快速检测方法，其特征在于，所述步骤(3)中，待测样本DNA的浓度需求＝5ng/uL×待测物种基因组大小/人类基因组大小。
  4. 如权利要求1或2所述的一种近交系遗传质量监控的SNP快速检测方法，其特征在于，所述KASP法的反应体系如下：包括浓度为5-50ng/uL的DNA在每个反应孔的体积为0.8uL，2×KASP Master mix在每个反应孔的体积为0.778uL，KASP Primer mix在每个反应孔的体积为0.022uL。
  5. 如权利要求1或2所述的一种近交系遗传质量监控的SNP快速检测方法，其特征在于，所述KASP法的反应程序如下：
    Figure PCTCN2019075790-appb-100001
    或
    Figure PCTCN2019075790-appb-100002
    或
    Figure PCTCN2019075790-appb-100003
  6. 一组位点在近交系遗传质量监控中作为SNP位点的应用，其特征在于，所述一组位点有96个，具体如下
    Figure PCTCN2019075790-appb-100004
    Figure PCTCN2019075790-appb-100005
    Figure PCTCN2019075790-appb-100006
    Figure PCTCN2019075790-appb-100007
    *为特异性区分位点。
  7. 一种近交系遗传质量监控的SNP位点引物，其特征在于，所述位点引物如SEQ ID NO.1至SEQ ID NO.288所示。
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