WO2016188144A1 - 一种具有增强鉴别能力的str基因座荧光标记复合扩增试剂盒及其应用 - Google Patents

一种具有增强鉴别能力的str基因座荧光标记复合扩增试剂盒及其应用 Download PDF

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WO2016188144A1
WO2016188144A1 PCT/CN2016/073351 CN2016073351W WO2016188144A1 WO 2016188144 A1 WO2016188144 A1 WO 2016188144A1 CN 2016073351 W CN2016073351 W CN 2016073351W WO 2016188144 A1 WO2016188144 A1 WO 2016188144A1
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seq
nos
kit
loci
amplification
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金海英
张兹钧
余丁
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宁波海尔施基因科技有限公司
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  • the invention relates to a PCR amplification kit, in particular to a fluorescent composite amplification kit for simultaneously detecting 24 autosomal loci, 2 Y chromosome loci and 1 sex locus in a single tube, and the invention also relates to The preparation method of the fluorescent composite amplification kit and the application of the kit in the field of forensic identification belong to the fields of autochromic and Y chromosome typing and identification.
  • a short tandem repeat is a type of DNA sequence with a length polymorphism formed by tandem repeats of 2-6 bases as a core unit in the human genome. The number of core units varies and the number of repeats is different. It constitutes the genetic polymorphism of STR. STR is widely distributed and multiplied, accounting for about 10% of the human genome. It contains a huge amount of information. Different sequences can produce hundreds of millions of genotype combinations, and each combination has a very low frequency in the population. It has a very high ability to identify individuals, so it is often used as a genetic marker for forensic individual identification and kinship identification in DNA analysis technology.
  • the FBI of the United States first selected 13 STR loci for DNA database - CODIS (Combined DNA Index System): D3S1358, TH01, D21S11, D18S51, D5S818, D13S317, D7S820, CSF1PO, vWA, D8S1179, TPOX, FGA, D16S539 .
  • STR loci are commonly referred to as 13 core loci, and the most prominent ones are the ABI's Identifiler kit and Promega's PowerPlex-16 Fluorescence Detection Kit, which contain the 13 core loci described above.
  • Patent CN101144774 (Human STRtyper PCR Amplification Fluorescence Detection Kit) discloses a fluorescently labeled complex amplification system for simultaneously analyzing multiple loci of human genomic DNA for detecting the following 21 autosomal loci: TPOX, D3S1358, D5S818, FGA, CSF1PO, D8S1179, D7S820, TH01, vWA, D13S317, D16S539, D18S51, D21S11, D12S391, D19S433, D1S1656, D2S1338, D6S1043, PentaE, PentaD, Amel.
  • STR locus fluorescent labeling complex amplification kit for enhanced discrimination.
  • the present invention is directed to the above technical problem, and provides a STR locus fluorescent labeling composite amplification kit with enhanced discrimination capability, which comprises all the loci used by various manufacturers at home and abroad for forensic identification and paternity testing.
  • the kit adds the four autosomal loci of SE33, D2S441, D10S1248 and D22S1045 commonly used in Europe and 21 loci Amil, which are commonly used in sex determination, under the 21 loci in CN101144774 (STRtyper-21G).
  • the chromosomal loci DYS391 and Y-indel can simultaneously amplify 27 loci in one reaction, which makes the database coverage reach a great coverage, which greatly saves the cost of reagents and labor, and improves the work efficiency.
  • the loci are D3S1358, TH01, D21S11, D18S51, Penta E, Y-indel, DYS391, D12S391, D6S1043, D2S1338, D1S1656, Amel, D5S818, D13S317, D7S820, D19S433, CSF1PO, Penta D, D2S441, vWA, D8S1179, TPOX, FGA, D16S539, D22S1045, SE33 and D10S1248.
  • the amplification system of the present invention includes a primer mixture, a reaction buffer, and the like.
  • primers were designed for the above 27 loci in the flank of their core repeat regions. Primers were designed using Primer 5 software, and the annealing temperature of each primer was around 60 °C. Primer dimers, other interactions or cross-reactions cannot be generated between the primers, and the length of the amplified product is between 70-500 bp. Amplification tests were performed on each pair of primers and optimized until a clear single amplified band was obtained.
  • the specific primer sequences are shown in Table 1 below.
  • the present invention marks the 5' end of one primer in each pair of primers by rational arrangement of individual loci and preferably a series of fluorescent dyes with high fluorescence intensity, and the labeling methods are as follows: D3S1358, TH01, D21S11, D18S51 and Penta E
  • Y-indel, DYS391, D12S391, D6S1043, D2S1338, and D1S1656 are the second group, using HEX label
  • D5S818, D13S317, D7S820, D19S433, CSF1PO, and Penta D are the third group, using TAM Marking
  • D2S441, vWA, D8S1179, TPOX and FGA are the fourth group, using ROX label
  • Amel, D16S539, D22S1045, SE33 and D10S1248 are the fifth group, using Alex 594 label
  • each locus was separated by the length of the fragment of the amplification primer, and the primer sequence was optimized so that there was no non-specific band in the amplification range, and then the primer concentration was adjusted so that the same fluorescein was between different loci.
  • the peak height balance is 50%, and the peak height balance between different fluoresceins is 30%.
  • the method for detecting an amplification product of the present invention is carried out by using a multi-channel or single-channel capillary electrophoresis genetic analyzer; the template determined by the present invention includes human blood, blood mark, semen, saliva, body fluid, hair, muscle or tissue organ, and Direct amplification filter paper, FTA card, cotton wool, buccal swab and other materials can be used.
  • the template determined by the present invention includes human blood, blood mark, semen, saliva, body fluid, hair, muscle or tissue organ, and Direct amplification filter paper, FTA card, cotton wool, buccal swab and other materials can be used.
  • the STR locus fluorescent labeling complex amplification kit with enhanced discrimination capability comprises nuclease-free water, PCR buffer, primer mixture, allelic standard, positive control, molecular weight internal standard and spectrum
  • a calibration standard which is a mixture of all of the different genotypes of the locus contained in the kit in a population of cells, the spectral calibration standards being fluorescent PCR amplification products of different sizes .
  • the STR locus fluorescent labeling composite amplification kit with enhanced discrimination capability further comprises nuclease-free water, PCR Master Mix, Primer Mix, Allelic ladder mixture and internal standard Size-500.
  • the PCR Master Mix used in the present invention has been subjected to a series of optimization experiments to make the product compatible with all common types of materials on the market including whatman FTA card, whatman saliva card, blood filter paper, Bokun FTA card, Bo Kun's saliva card, hair, oral exfoliated cells, DNA extraction and other samples, which have not been done in domestic or even foreign kits, in addition, this improved buffer can greatly improve the amplification efficiency
  • the invention can effectively shorten the time of adenylation at the end of the product, shorten the overall amplification time, and can improve the amplification efficiency of the long fragment and improve the balance of the product.
  • Its main components are: DMSO, Tris-buffer, potassium chloride, ammonium sulfate and dNTP (dATP, dG
  • the enzyme required for the amplification reaction is a hot-start DNA polymerase, which can be modified by an antibody or chemically modified, and an amount of 2U-4U of enzyme is required in the reaction system.
  • the amplification system is carried out in various reaction thermal cyclers (such as ABI9700, ABI9600, Bio-Rad S1000, etc.), using the following procedure: incubation at 95 ° C for 2-10 minutes; incubation at 94 ° C for 5-10 seconds, insulation at 61 ° C 60 seconds, 70 ° C insulation 30-60 seconds, this step runs 28 cycles; 60 ° C insulation 15-30min, 4 ° C insulation.
  • reaction thermal cyclers such as ABI9700, ABI9600, Bio-Rad S1000, etc.
  • the amplification product since fluorescently labeled primers are used, the amplification product also carries a fluorescent label, and the label can emit an optical signal that can be recognized by a genetic analyzer (such as ABI3500, 3500genetic analyzer) under laser excitation, so the amplification product Electrophoresis and detection analysis can be performed in instruments such as genetic analyzers.
  • a genetic analyzer such as ABI3500, 3500genetic analyzer
  • the amplified product When tested in a genetic analyzer, the amplified product is mixed with a molecular weight internal standard (Size-500) and formamide in a certain ratio, and enters the instrument capillary or gel for electrophoretic separation.
  • the molecular weight internal standard is composed of a plurality of fluorescent-labeled DNA fragments of known length, which are used to calculate the length of the PCR amplification product fragment, so that genotyping and alignment with the allelic ladder can be judged.
  • the post-electrophoresis data can be analyzed on the GeneMapper ID-X data analysis software to obtain STR genotyping profiles and data.
  • the invention selects six-color fluorescence technology after screening and testing different fluorescein, and has a qualitative breakthrough than the five-color fluorescence technology generally popular in the market.
  • the kit provided by the invention has more detection loci than the similar products on the market at home and abroad, thereby greatly increasing the cumulative individual recognition ability and the cumulative non-parent exclusion rate of the system, and generally improving the discriminating power of the individual.
  • the two Y chromosome loci added can assist in gender determination, especially in the case of male Y chromosome sex gene deletion, which can be used to determine gender, which makes the kit more practical and functional.
  • Table 2 below compares the locus information of the present invention with the domestic market multi-locus mainstream kit:
  • Table 2 Comparison of the locus information of the present invention (SureID PanGlobal) and the current mainstream multi-locus kits in the domestic market
  • Figure 1 shows the Control DNA 9948 sample map.
  • Figure 2 is a map of the allelic typing standard.
  • Figure 3-a shows the typing map of the father being tested.
  • Figure 3-b shows the classification map of the child being examined.
  • Figure 4 is a partial map of a male Y chromosome sex gene deletion sample amplified using the kit of the present invention.
  • Figure 5 is a typing map of a male Y chromosome sex gene deletion sample amplified using the PowerPlex 18D kit.
  • the invention has identified and selected fluorescent dyes, and selected six fluorescent labels of blue, green, yellow, red, purple and orange to construct a six-color fluorescent combination scheme. Based on the determination of the 6-color fluorescence combination scheme, the gene locus combination method and the fluorescent label type were designed through a large number of repeated experiments. In terms of production cost and amplification efficiency of primers at each locus, 27 loci were divided into 5 groups, using FAM, HEX, TAM, ROX, and Alex 594 group markers, and the molecular weight internal standard was the sixth color orange fluorescent dye. Atto 633 is marked.
  • a preferred method for labeling fluorescent dyes that has been finalized by screening is: D3S1358, TH01, D21S11, D18S51, and Penta E as the first group, using FAM labeling; Y-indel, DYS391, D12S391, D6S1043, D2S1338, and D1S1656 as the first Two groups, using HEX label; D5S818, D13S317, D7S820, D19S433, CSF1PO and Penta D as the third group, using TAM label; D2S441, vWA, D8S1179, TPOX and FGA as the fourth group, using ROX mark; Amel, D16S539, As the fifth group, D22S1045, SE33 and D10S1248 were labeled with Alex 594; the internal standard was selected with orange fluorescent label and the fluorescent label was Atto 633. This combination of loci allows simultaneous detection and analysis of 27 loci with only 6 fluorescent labels.
  • the PCR Master Mix includes: DMSO 10 mM, Tris-buffer 125 mM, potassium chloride 125 mM, ammonium sulfate 65 mM, deoxynucleotide triphosphate (dATP, dGTP, dTTP, dCTP) 7.5 mM, BSA 2.5 mg/ml, which can be achieved. Compatible with a wide range of common materials in the market.
  • the Primer Mix included all primers that amplify 27 loci (see Table 3 for concentration), 2-4 U/6.25 ⁇ l of Taq enzyme, and 7.5 mM magnesium chloride.
  • Control DNA 9948 was used as a positive control and was human genomic DNA purchased from Suzhou Xinhai Biotechnology Co., Ltd.
  • the Allelic Ladder allelic ladder is a mixture of all the different genotypes of the locus contained in the kit in a certain population, from Ningbo Haiershi Gene Technology Co., Ltd.
  • the Size-500 orange fluorescent molecular weight internal standard is a series of amplification products for calibrating a certain fragment size, from Ningbo Haiershi Gene Technology Co., Ltd.
  • the spectral calibration standard is a fluorescent PCR amplification product of six different size fragments from Ningbo Haiershi Gene Technology Co., Ltd.
  • step temperature time 1 95 ° C 2-10 minutes 2 94°C 5-10 seconds 3 61 ° C 1 minute 4 70 ° C 30-60 seconds 5 N/A Repeat 2-4 steps 27 times (28 times in total) 6 60 ° C 15-30 minutes 7 4 ° C Continuous: until the PCR product is collected
  • a loading mixture (25-50 ⁇ L Size-500 + 1000 ⁇ L deionized formamide) was composed of deionized formamide and a molecular weight internal standard (Size-500) in the system.
  • the analysis was carried out by genetic analysis instrument. The specific analysis parameters were injection voltage: 1.2kv, injection time: 15s, electrophoresis time 1210-1500s.
  • Sensitivity analysis After the positive control is diluted by a certain copy number ratio, it is detected by PCR amplification and capillary electrophoresis until no signal is detected.
  • the copy number is the lowest detection line, which is the sensitivity of the kit. The highest sensitivity detects DNA samples as low as 0.125 ng.
  • the fluorescent labeling complex amplification test system of 27 loci in the present invention tests pigs, dogs, sheep, ducks, chickens, mice, cows, E. coli, etc., without specific amplification peaks, indicating that the system has Species specificity.
  • the kit provided by the invention is used for the paternity test of the relationship between the father and the child to be tested, and the measurement steps are as follows:
  • the sample in this case is a filter paper blood sample, which is directly amplified, so it is only necessary to use a 1.2mm puncher for punching as a test template.
  • Amplification detection According to Examples 2 to 4, fluorescent labeling, PCR amplification, and genetic analyzer detection were carried out, and a kit for amplifying a primer pair of a specific oligonucleotide comprising 27 loci of the present invention was selected. The results of the father's classification are shown in Figure 3-a, and the child's classification results are shown in Figure 3-b. The comparison results are shown in the following table:
  • the paternity test in the paternity test, because one parent cannot provide genetic information, the probability of his parentality is relatively low relative to the triplet detection. Therefore, in order to ensure the accuracy of the paternity test, it is usually necessary to add more loci. In the paternity test of the single parent case, if a contradiction locus is found, it should be increased to more than 26 loci. If there are 3 or more contradictory loci, the conclusion of "negative birth relationship" can be made.
  • the kit provided by the invention is used for sex identification under the deletion of the sex locus on the Y chromosome of the male sample, and the determination steps are as follows:
  • Samples are provided by a public security bureau.
  • the sample in this case is a filter paper blood sample, which is directly amplified, so it is only necessary to use a 1.2 mm puncher for punching as a test template.
  • Amplification detection According to Examples 2 to 4, fluorescent labeling, PCR amplification, and genetic analyzer detection were carried out, and a kit for amplifying a primer pair of a specific oligonucleotide comprising 27 loci of the present invention was selected. The classification map is shown in Figure 4. At the same time, the sample is amplified simultaneously with PowerPlex 18D. The classification map is shown in Figure 5. The classification results are shown in Table 5 below:
  • the tested sample owner has normal male signs and is determined to be a male sample.
  • the laboratory uses the PowerPlex18D kit to detect that the gender gene is only X, which is a female sample.
  • the test results of the kit of the present invention showed that although the Amel locus has only X, there are specific target bands at both Y-indel and DYS391 loci, so it can be determined that the sample is a male sample.
  • the Amel locus is located at the short arm of the Y chromosome, which is more likely to cause deletion.
  • Y-indel and DYS391 are located on the long arm of the Y chromosome, which can greatly reduce the risk of sex determination in the absence of sex loci. If you use the common forensic typing test kit currently available on the market, only the Amel locus, if you encounter the Y chromosome deletion of the locus, it is easy to determine the sample as a female sample. If it is an unknown gender sample in the case, it will completely affect the direction and progress of the case. If it is encountered in the batch construction, it will find that the gender does not match when checking the gender, it will also cause great trouble to a large number of database construction work. In either case, it will cause great waste of manpower, material resources and time. With the kit of the invention, the sex of the sample in such cases can be accurately determined, and manpower, material resources and time are greatly saved.

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Abstract

提供了一种具有增强鉴别能力的STR基因座荧光标记复合扩增试剂盒，所述试剂盒同时扩增27个STR基因座；包括下列24个常染色体STR基因座：D3S1358、TH01、D21S11、D18S51、Penta E、D12S391、D6S1043、D2S1338、D1S1656、D2S441、D5S818、D13S317、D7S820、D19S433、CSF1PO、Penta D、vWA、D8S1179、TPOX、FGA、D16S539、D22S1045、SE33和D10S1248，2个Y染色体基因座Y-indel、DYS391及一个性别决定位点Amel。

Description

一种具有增强鉴别能力的STR基因座荧光标记复合扩增试剂盒及其应用 技术领域
本发明涉及一种PCR扩增试剂盒，尤其涉及一种单管同时检测24个常染色体基因座、2个Y染色体基因座、1个性别基因座的荧光复合扩增试剂盒，本发明还涉及该荧光复合扩增试剂盒的制备方法以及该试剂盒在司法鉴定领域中的应用，属于常染色体和Y染色体分型和鉴定领域。
背景技术
短串联重复序列(short tandem repeat，STR)是人类基因组中由2-6个碱基作为核心单位串联重复形成的一类具有长度多态性的DNA序列，其核心单位的数目变化和重复次数不同构成了STR的遗传多态性。STR分布广，数目多，约占人类基因组的10％，所包含的信息量巨大，不同的序列可以产生数以亿计的基因型组合，而每一种组合在群体中出现的频率都非常低，具有极高的个体鉴定能力，所以常作为遗传标记而被用于DNA分析技术中法医个体识别、亲缘关系鉴定。随着这一技术的发展，很多国家利用该项技术建立了犯罪分子及嫌疑人的DNA数据库，也就是对犯罪嫌疑人的DNA数据进行分析并保存到数据库，便于进行比对和排查等工作项目。同时也是DNA数据库建立的主流技术。同时，STR基因座的片段小，容易扩增，适宜于检验微量和降解检材，而且各基因座的扩增条件相似而能够复合扩增，因而具有灵敏、准确、快速、信息量大等优点。由于这些优点STR基因座的分型研究与筛选早已在国内外人类学、医学遗传学和法医学及各相关领域中得到了广泛的应用。
美国FBI最早选择了13个STR基因座用于建立DNA数据库——CODIS(Combined DNA Index System)：D3S1358、TH01、D21S11、D18S51、D5S818、D13S317、D7S820、CSF1PO、vWA、D8S1179、TPOX、FGA、D16S539。这些STR基因座通常被称为13个核心基因座，目前最有代表的是美国ABI公司的Identifiler试剂盒和Promega公司的PowerPlex-16荧光检测试剂盒，均包含了上述的13个核心基因座。但是随着DNA作为鉴定手段的应用越来越广泛，用户对试剂盒的基因座数目、信息量、扩增时间、检材的适用范围等有了越来越高的要求，并且随着我国DNA数据库建设规模的不断扩大，数据比对的作用也日益重要，由于数据比对建立在相同的STR基因座上，因此也需要STR试剂盒在基因座上具有兼容性，否则会导致DNA数据库中部分数据资源浪费。
专利CN101144774(人类STRtyper PCR扩增荧光检测试剂盒)公开了一种同时分析人类基因组DNA的多个基因座的荧光标记复合扩增系统，用于检测如下21个常染色体基因座：TPOX、D3S1358、D5S818、FGA、CSF1PO、D8S1179、D7S820、TH01、vWA、D13S317、D16S539、D18S51、D21S11、D12S391、D19S433、D1S1656、D2S1338、D6S1043、PentaE、PentaD、Amel。
为了减少检测时间，提高试剂盒的个体识别能力和累计非父排除率，使其能覆盖全球大多数国家的DNA数据库，并同时节约试剂和人力成本、提高工作效率，需要研发一种新的具有增强鉴别能力的STR基因座荧光标记复合扩增试剂盒。
发明内容
本发明针对上述技术问题，提供了一种具有增强鉴别能力的STR基因座荧光标记复合扩增试剂盒，其包含了目前国内外各个生产商所采用的全部基因座，用于法医鉴定及亲子鉴定。该试剂盒在传承了CN101144774(STRtyper-21G)中21个基因座下添加了欧洲常用的SE33、D2S441、D10S1248、D22S1045这四个常染色体基因座和一个辅助性别判定的基因座Amel，两个Y染色体基因座DYS391和Y-indel，可以同时在一次反应中扩增27个基因座，使数据库排查达到了极大的覆盖率，大大节约了试剂和人力的成本，同时提高了工作效率。
所述的基因座为D3S1358、TH01、D21S11、D18S51、Penta E、Y-indel、DYS391、D12S391、D6S1043、D2S1338、D1S1656、Amel、D5S818、D13S317、D7S820、D19S433、CSF1PO、Penta D、D2S441、vWA、D8S1179、TPOX、FGA、D16S539、D22S1045、SE33和D10S1248。
本发明的扩增体系包括引物混合物、反应缓冲液等。
首先针对上述27个基因座在其核心重复序列区的侧翼分别设计特异性引物。采用Primer 5软件设计引物，每条引物的退火温度在60℃左右。引物之间不能产生引物二聚体、其它相互作用或交叉反应，并且扩增产物的长度在70-500bp之间。对每对引物进行扩增测试并优化，直至得到清晰单一的扩增条带。具体引物序列见下表1。
表1：各基因座对应的引物序列及其浓度
Figure PCTCN2016073351-appb-000001
Figure PCTCN2016073351-appb-000002
Figure PCTCN2016073351-appb-000003
本发明通过对各个基因座的合理排列并优选了一系列荧光强度高的荧光染料对每对引物中的一条引物的5’端进行标记，标记方法如下：D3S1358、TH01、D21S11、D18S51和Penta E为第一组，采用FAM标记；Y-indel、DYS391、D12S391、D6S1043、D2S1338和D1S1656为第二组，采用HEX标记；D5S818、D13S317、D7S820、D19S433、CSF1PO和Penta D为第三组，采用TAM标记；D2S441、vWA、D8S1179、TPOX和FGA为第四组，采用ROX标记；Amel、D16S539、D22S1045、SE33及D10S1248为第五组，采用Alex 594标记；内标选用橙色荧光标记，荧光标记物为Atto 633。每组中通过扩增引物的片段长短将每个基因座分开，并优化引物序列使扩增范围内不存在非特异条带，然后调整引物浓度以使同一个荧光素的不同基因座之间的峰高均衡性达到50％，不同荧光素之间的峰高均衡性达到30％，其中对Y-indel和DYS391这两个Y染色体基因座没有均衡性要求，但是需要在0.125ng的阳性对照下能够进行准确的分型。
本发明检测扩增产物的方法为采用多道或单道毛细管电泳遗传分析仪进行测定；本发明所测定的模板包括人的血液、血痕、精液、唾液、体液、毛发、肌肉或组织器官，并且可以进行直接扩增滤纸、FTA卡、棉絮、口腔拭子等检材。
本发明提供的具有增强鉴别能力的STR基因座荧光标记复合扩增试剂盒包括无核酸酶水、PCR缓冲液、引物混合液、等位基因分型标准品、阳性对照品、分子量内标以及光谱校正标准物，所述等位基因分型标准品是试剂盒所包含的基因座在一定数量人群中的所有不同基因分型的混合物，所述光谱校正标准物是不同大小的荧光PCR扩增产物。
本发明提供的具有增强鉴别能力的STR基因座荧光标记复合扩增试剂盒还包括无核酸酶水、PCR Master Mix、Primer Mix、Allelic ladder混合物、内标Size-500。值得一提的是本发明所采用的PCR Master Mix经过一系列的优化实验使得本产品可以兼容市面上所有常见的检材类型包括whatman FTA卡、whatman唾液卡、血滤纸、博坤FTA卡、博坤唾液卡、头发、口腔脱落细胞、提取DNA等各种检材，这在国内甚至国外的试剂盒也尚未做到，除此之外，该种改良后的缓冲液能极大地提高扩增效率，有效缩短产物末端腺苷酰化的时间，缩短整体扩增时间，并能够提高长片段的扩增效率，改善了产品的均衡性。 其主要成分有：DMSO、Tris-buffer、氯化钾、硫酸铵和dNTP(dATP、dGTP、dTTP、dCTP)等。
扩增反应需要的酶为热启动DNA聚合酶，所述酶可经过抗体修饰或化学修饰，本反应体系中需要加入2U-4U的酶量。
扩增体系在各种反应热循环仪(如ABI9700、ABI9600、Bio-Rad S1000等)中进行，采用以下的程序：95℃保温2-10分钟；94℃保温5-10秒钟，61℃保温60秒钟，70℃保温30-60秒钟，该步骤运行28个循环；60℃保温15-30min，4℃保温。
本发明由于采用了荧光标记的引物，扩增产物也带有荧光标记物，并且标记物可以在激光激发下发出可通过遗传分析仪(如ABI3500、3500genetic analyzer)识别的光信号，所以扩增产物可以在遗传分析仪等仪器中进行电泳和检测分析。
在遗传分析仪中进行检测时，扩增产物与分子量内标(Size-500)、甲酰胺按照一定比例混合，进入仪器毛细管或凝胶中进行电泳分离。分子量内标是由多条已知长度的荧光标记DNA片段组成，用来计算PCR扩增产物片段长度，从而可以判断基因分型以及与等位基因阶梯比对。
电泳后的数据可以在GeneMapper ID-X数据分析软件上分析，得到STR基因分型图谱和数据。
本发明的有益效果：
本发明在传承了CN101144774(STRtyper-21G)21个基因座下添加了欧洲常用的SE33、D2S441、D10S1248、D22S1045四个常染色体基因座和辅助性别判定基因座Amel，以及两个Y染色体基因座DYS391和Y-indel，可以同时在一次反应中扩增27个基因座，使数据库排查达到了极大的覆盖率，大大节约了试剂和人力的成本，同时提高了工作效率。
本发明在筛选试用了不同的荧光素后，选择了六色的荧光技术，比市面普遍流行的五色荧光技术有了质的突破。本发明所提供的试剂盒的检测基因座数量多于市面上国内外的同类产品，从而极大地提高了系统的累积个体识别力和累积非父排除率，总体上提高了个体的鉴别力。同时所添加的两个Y染色体基因座能辅助性别判定，尤其在男性Y染色体性别基因缺失的情况下，可用于判定性别，使得试剂盒的实用性和功能性大增。下表2对本发明与国内市场多基因座主流试剂盒的基因座信息进行了比较：
表2：本发明(SureID PanGlobal)与目前国内市场多基因座主流试剂盒的基因座信息的比较
Figure PCTCN2016073351-appb-000004
注：+表示该基因座包含，-表示不包含
附图说明
图1为Control DNA 9948样本图谱。
图2为等位基因分型标准品图谱。
图3-a为被检父亲的分型图谱。
图3-b为被检孩子的分型图谱。
图4为男性Y染色体性别基因缺失样本用本发明的试剂盒扩增的分型图谱。
图5为男性Y染色体性别基因缺失样本用PowerPlex 18D试剂盒扩增的分型图谱。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
实施例1基因座的确定
在本公司上个产品(人类STRtyper-21G扩增荧光检测试剂盒)的基础上添加了欧洲常用的SE33、D2S441、D10S1248、D22S1045四个常染色体基因座和一个辅助性别判定的基因座Amel，以及两个Y染色体基因座DYS391和Y-indel，最后确定D3S1358、TH01、D21S11、D18S51、Penta E、Y-indel、DYS391、D12S391、D6S1043、D2S1338、D1S1656、D5S818、D13S317、D7S820、D19S433、CSF1PO、Penta D、D2S441、vWA、D8S1179、TPOX、FGA、Amel、D16S539、D22S1045、SE33和D10S1248这27个基因座。
实施例2荧光标记复合扩增体系的基因座组合方案设计
本发明对荧光染料进行了鉴别、遴选，选用了蓝、绿、黄、红、紫、橙六种荧光标记物，构建了6色荧光组合方案。在确定6色荧光组合方案的基础上，通过大量反复实验，设计出基因座组合方式以及荧光标记类型。从生产成本及各基因座引物扩增效率等方面考虑，将27个基因座分成5组，使用FAM、HEX、TAM、ROX及Alex 594分组标记，分子量内标用第6种颜色橙色的荧光染料Atto 633进行标记。经过筛选最终确定的一种优选的荧光染料标记的方法为：D3S1358、TH01、D21S11、D18S51和Penta E作为第一组，采用FAM标记；Y-indel、DYS391、D12S391、D6S1043、D2S1338和D1S1656作为第二组，采用HEX标记；D5S818、D13S317、D7S820、D19S433、CSF1PO和Penta D作为第三组，采用TAM标记；D2S441、vWA、D8S1179、TPOX和FGA作为第四组，采用ROX标记；Amel、D16S539、D22S1045、SE33及D10S1248作为第五组，采用Alex 594标记；内标选用橙色荧光标记，荧光标记物为Atto 633。这种基因座组合方式使得仅需标记6种荧光就可以实现27个基因座同时检测分析。
实施例3扩增基因座及其产物检测的实验过程
本发明提供的具有增强鉴别能力的STR基因座荧光标记复合扩增试剂盒包括：
1)PCR Master Mix
2)Primer Mix
3)Control DNA 9948
4)Allelic Ladder等位基因分型标准品
5)Size-500橙色荧光分子量内标
6)光谱校正标准物
所述PCR Master Mix包括：DMSO 10mM、Tris-buffer 125mM、氯化钾125mM、硫酸铵65mM、三磷酸脱氧核苷酸(dATP、dGTP、dTTP、dCTP)7.5mM、BSA 2.5mg/ml，可以实现兼容扩增市面常见各种检材。
所述Primer Mix包括扩增27个基因座的所有引物(浓度见表3)，Taq酶2-4U/6.25μl、氯化镁7.5mM。
所述Control DNA 9948作为阳性对照品，是人类基因组DNA，购自苏州新海生物科技股份有限公司。
所述Allelic Ladder等位基因分型标准品是试剂盒所包含的基因座在一定数量人群中的所有不同基因分型的混合物，来自于宁波海尔施基因科技有限公司。
所述Size-500橙色荧光分子量内标是一系列用于标定一定片段大小的扩增产物，来自宁波海尔施基因科技有限公司。
所述光谱校正标准物是6种不同大小片段的荧光PCR扩增产物，来自宁波海尔施基因科技有限公司。
1、反应体系的配置
组分 体积
无核酸酶水(无RNA酶/DNA酶超纯水Dnase/Rnase Free) 补足至25.0μL
PCR Master Mix 12.5μL
PrimerMix 6.25μL
样本DNA 0.125-2ng
总体积 25μL
2、扩增热循环实验方案
1)将PCR扩增管置于热循环仪上；
2)选择下表4推荐的程序进行扩增。
3)扩增后的样品应避光保存；
表4：热循环仪的扩增程序
步骤 温度 时间
1 95℃ 2-10分钟
2 94℃ 5-10秒钟
3 61℃ 1分钟
4 70℃ 30-60秒钟
5 N/A 重复2-4步骤27次(共28次)
6 60℃ 15-30分钟
7 4℃ 持续:直至收取PCR产物
3、扩增产物在遗传分析仪上荧光检测
由去离子甲酰胺与系统中分子量内标(Size-500)组成上样混合物(25-50μL Size-500+1000μL去离子甲酰胺)。将9μL上样混合物与1μL扩增产物或系统中等位基因分型标准物(Allelic ladder)混合，避免产生气泡，尽快电泳。用遗传分析仪检测分析，具体分析参数为进样电压：1.2kv，进样时间：15s，电泳时间1210-1500s。
实施例4检测试剂盒灵敏度、特异性分析
灵敏度分析：将阳性对照按一定拷贝数倍比稀释后，经PCR扩增和毛细管电泳检测直至检测不到信号，该拷贝数即为最低检测线，也就是试剂盒的灵敏度。最高灵敏度可检测到低至0.125ng的DNA样品。
特异性分析：本发明中27个基因座的荧光标记复合扩增检验系统检验猪、狗、羊、鸭、鸡、鼠、牛、大肠杆菌等，没有出现特异性扩增峰，表明该系统具有种属特异性。
实施例5本发明所提供的试剂盒在亲子鉴定中的应用
将本发明所提供的试剂盒用于被检父亲与孩子关系的亲子鉴定，测定步骤如下：
1.收集亲子鉴定案件中的血斑：亲子鉴定样本由某司法鉴定所提供
2.被检检材的处理：本案中的检材为滤纸血样，采用直接扩增，故只需使用1.2mm的打孔器进行打孔作为检测模板
3.扩增检测：按照实施例2～4进行荧光标记、PCR扩增和遗传分析仪检测，选用本发明的包含27个基因座的特异性寡核苷酸扩增引物对的试剂盒，被检父亲的分型结果见图3-a，孩子的分型结果见图3-b，其对比结果见下表：
Figure PCTCN2016073351-appb-000005
注：下划线表示不符合遗传规律的基因座
结论：
结果显示，被检男孩与被检父在vWA、D12S391和SE33这三个基因座上不符合遗传规律，排除他们的亲子关系。
二联体检测在亲子鉴定中由于亲代某一方不能提供遗传信息，因此导致其亲权的概率相对三联体检测较低，所以为了保证亲子鉴定的准确性，通常都需要增加更多的基因座。在单亲案的亲子鉴定中，如果发现一个矛盾基因座，就要增加到26个基因座以上，如果出现3个或3个以上矛盾基因座，则可做出“否定亲生关系”的结论。
在本实施例中，我司选用了PowerPlex 18D试剂盒(购自Promega公司)检测了17个基因座，发现被检男孩在vWA这一个基因座上不符合遗传规律，不能排除亲生关系。用我司上一代产品STRtyper-21G时发现有vWA和D12S391两个基因座不符合遗传规律，依旧不能排除亲生关系。若将检测的基因座再增加几个，可以明显提高非父排除率。用本发明的试剂盒同时检测27个基因座发现，本实施例中的男孩同时有3个基因座违反遗传规律，则可以做出“否定亲生”的结论。
实施例6本发明所提供的试剂盒在男性Y染色体性别基因缺失下的性别鉴定中的应用
将本发明所提供的试剂盒用于男性样本Y染色体上性别基因座缺失下的性别鉴定，测定步骤如下：
1.收集男性样本的血斑：样本由某公安局提供。
2.被检检材的处理：本案中的检材为滤纸血样，采用直接扩增，故只需使用1.2mm的打孔器进行打孔作为检测模板。
3.扩增检测：按照实施例2～4进行荧光标记、PCR扩增和遗传分析仪检测，选用本发明的包含27个基因座的特异性寡核苷酸扩增引物对的试剂盒，其分型图谱见图4，同时选用PowerPlex 18D同时扩增该样本，分型图谱见图5，分型结果见下表5：
表5：本发明的试剂盒与PowerPlex 18D试剂盒检测该男性样本的结果
Figure PCTCN2016073351-appb-000006
Figure PCTCN2016073351-appb-000007
注：-表示无该基因座
4.结论：
被检测的样本主人有着男性正常的体征，确定为男性样本，本实验室选用PowerPlex18D试剂盒检测后结果显示性别基因只有X，为女性样本。用本发明的试剂盒检测结果显示虽然Amel基因座只有X，但是在Y-indel和DYS391这两个基因座上均有特异性的目的条带，因此可以判定该样本为男性样本。
Amel基因座位于Y染色体短臂处，较易造成缺失，而Y-indel和DYS391位于Y染色体长臂处，可以将性别基因座缺失下的性别判定风险大大降低。如果用目前市面上常见的法医分型检测试剂盒，只有Amel基因座，一旦遇到该基因座Y染色体缺失，就容易将该样本判定成女性样本。如果是案件中未知的性别样本，将完全影响案件的方向和进度，如果是在批量建库中遇到，在核对男女性别时发现性别不符，也会对大量的建库工作造成极大的困扰，不管是哪种情况，都会造成极大的人力、物力和时间的浪费，而使用本发明的试剂盒，可以准确地判定该类情况下的样本性别，极大地节约人力、物力和时间。
上述实施例并非对本发明的限制，本发明也并不限于上述实施例。本技术领域的普通 技术人员在本发明的实质范围内，做出的变化、改型、添加或替换，也应属于本发明的保护范围。

Claims (10)

  1. 一种具有增强鉴别能力的STR基因座荧光标记复合扩增试剂盒，其包括下列27个基因座的特异性寡核苷酸扩增引物对：D3S1358、TH01、D21S11、D18S51、Penta E、D12S391、D6S1043、D2S1338、D1S1656、D2S441、D5S818、D13S317、D7S820、D19S433、CSF1PO、Penta D、vWA、D8S1179、TPOX、FGA、D16S539、D22S1045、SE33、D10S1248、Y-indel、DYS391和Amel。
  2. 权利要求1的试剂盒，其中所述27个STR基因座对应的引物对如下：D3S1358：SEQ ID NO：1～2；TH01：SEQ ID NO：3～4；D21S11：SEQ ID NO：5～6；D18S51：SEQ ID NO：7～8；PENTAE：SEQ ID NO：9～10；Y-indel：SEQ ID NO：11～12；DYS391：SEQ ID NO：13～14；D12S391：SEQ ID NO：15～16；D6S1043：SEQ ID NO：17～18；D2S1338：SEQ ID NO：19～20；D1S1656：SEQ ID NO：21～22；D5S818：SEQ ID NO：23～24；D13S317：SEQ ID NO：25～26；D7S820：SEQ ID NO：27～28；D19S433：SEQ ID NO：29～30；CSF1PO：SEQ ID NO：31～32；PENTAD：SEQ ID NO：33～34；D2S441：SEQ ID NO：35～36；vWA：SEQ ID NO：37～38；D8S1179：SEQ ID NO：39～40；TPOX：SEQ ID NO：41～42；FGA：SEQ ID NO：43～44；Amel：SEQ ID NO：45～46；D16S539：SEQ ID NO：47～48；D22S1045：SEQ ID NO：49～50；SE33：SEQ ID NO：51～52；D10S1248：SEQ ID NO：53～54。
  3. 权利要求1的试剂盒，其中每对引物中有一条引物的5’端用荧光染料标记。
  4. 权利要求3的试剂盒，其中使用所述荧光染料进行标记的方法为：D3S1358、TH01、D21S11、D18S51和Penta E作为第一组，采用蓝色荧光染料FAM标记；Y-indel、DYS391、D12S391、D6S1043、D2S1338和D1S1656作为第二组，采用绿色荧光染料HEX标记；D5S818、D13S317、D7S820、D19S433、CSF1PO和Penta D作为第三组，采用黄色荧光染料TAM标记；D2S441、vWA、D8S1179、TPOX和FGA作为第四组，采用红色荧光染料ROX标记；Amel、D16S539、D22S1045、SE33和D10S1248作为第五组，采用紫色荧光染料Alex 594标记。
  5. 权利要求1的试剂盒，其中所述试剂盒设有内标，所述内标是不同大小的扩增产物。
  6. 权利要求5的试剂盒，其中所述内标用橙色荧光染料Atto 63标记。
  7. 权利要求1-6中任一项的试剂盒，其中所述试剂盒还包括如下组分：无核酸酶水、PCR 缓冲液、阳性对照品、等位基因分型标准品和光谱校正标准物；
    其中，所述无核酸酶水是指无RNA酶和DNA酶的超纯水，所述阳性对照品为人类基因组DNA，所述等位基因分型标准品是试剂盒所包含的所有基因座在一定数量人群中的所有不同基因分型的混合物，所述光谱校正标准物是不同大小的荧光PCR扩增产物。
  8. 权利要求7的试剂盒，其中所述PCR缓冲液包括DMSO 10mM、Tris-buffer 125mM、氯化钾125mM、硫酸铵65mM、三磷酸脱氧核苷酸(dATP、dGTP、dTTP、dCTP)7.5mM、BSA2.5mg/ml。
  9. 权利要求7的试剂盒，其中所述引物以引物混合液形式提供，所述引物混合液包括Taq酶2-4U/6.25μl、氯化镁7.5mM、以及浓度如下的同时扩增27个基因座的所有引物：
    SEQ ID NO：1～2：0.84μM；SEQ ID NO：3～4：0.5μM；SEQ ID NO：5～6：0.67μM；SEQ ID NO：7～8：0.66μM；SEQ ID NO：9～10：0.67μM；SEQ ID NO：11～12：1.34μM；SEQ ID NO：13～14：0.54μM；SEQ ID NO：15～16：1.26μM；SEQ ID NO：17～18：0.3μM；SEQ ID NO：19～20：0.7μM；SEQ ID NO：21～22：1.36μM；SEQ ID NO：23～24：0.5μM；SEQ ID NO：25～26：0.6μM；SEQ ID NO：27～28：0.65μM；SEQ ID NO：29～30：1.3μM；SEQ ID NO：31～32：1.2μM；SEQ ID NO：33～34：1.3μM；SEQ ID NO：35～36：0.65μM；SEQ ID NO：37～38：4.6μM；SEQ ID NO：39～40：1.8μM；SEQ ID NO：41～42：2.7μM；SEQ ID NO：43～44：1.9μM；SEQ ID NO：45～46：1.5μM；SEQ ID NO：47～48：2.0μM；SEQ ID NO：49～50：2.1μM；SEQ ID NO：51～52：2.3μM；SEQ ID NO：53～54：2.5μM。
  10. 权利要求1-9中任一项的试剂盒在性别鉴定、亲子鉴定或法医鉴定中的应用。
PCT/CN2016/073351 2015-05-28 2016-02-03 一种具有增强鉴别能力的str基因座荧光标记复合扩增试剂盒及其应用 WO2016188144A1 (zh)

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