WO2016070550A1 - 肌张力障碍vps16基因的检测引物、方法和试剂盒 - Google Patents

肌张力障碍vps16基因的检测引物、方法和试剂盒 Download PDF

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WO2016070550A1
WO2016070550A1 PCT/CN2015/075017 CN2015075017W WO2016070550A1 WO 2016070550 A1 WO2016070550 A1 WO 2016070550A1 CN 2015075017 W CN2015075017 W CN 2015075017W WO 2016070550 A1 WO2016070550 A1 WO 2016070550A1
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vps16
dystonia
gene
detecting
primer
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PCT/CN2015/075017
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French (fr)
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李维平
蔡晓东
吴松
刘文兰
张协军
陈欣
何思捷
刘欢
张建国
徐讯
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深圳市第二人民医院
深圳华大基因科技有限公司
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  • the invention belongs to the technical field of molecular bioengineering, and particularly relates to a detection primer, method and kit for the dystonia VPS16 gene.
  • Dystonia is the third most common dyskinesia disease following idiopathic tremor and Parkinson's disease. Its clinical manifestations are involuntary movements and postures caused by uncoordinated contraction of active muscles and antagonist muscles, or excessive contraction. Characteristics of sex and continuity. At present, in the research on dystonia in China, the pathogenic genes such as TOR1A, THAP1, GCH1, etc. have been found, but only less than 5% of cases can be explained; therefore, the detection of existing disease-causing genes cannot accurately identify the suspected Whether the sick patient is sick or not is accurately judged.
  • the object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a detection primer, method and kit for overcoming the one-sided dystonia VPS16 gene of the existing TOR1A, THAP1, GCH1 and the like mutation detection.
  • a detection primer for a dystonia VPS16 gene which comprises an upstream primer P1 having a nucleotide sequence of SEQ. ID. No. 1 of the Sequence Listing and a downstream primer having a nucleotide sequence of SEQ. ID. No. 2 of the Sequence Listing P2.
  • the above primer of the invention can realize the direct amplification of the standard DNA to obtain the VPS16 gene for analysis and judgment, overcome the one-sidedness of the mutation detection of the existing TOR1A, THAP1, GCH1, etc., and the process can be directly realized by the ordinary PCR method, It is fast, accurate, efficient, simple, and has a high early diagnosis rate.
  • the test results can provide scientific assistance for the early diagnosis, differential diagnosis and development of dystonia drugs for the treatment of dystonia.
  • the invention further proposes a method for detecting using the above primers, comprising the following steps:
  • the DNA standard to be tested is subjected to PCR amplification by an upstream primer P1 having a nucleotide sequence of SEQ. ID. No. 1 of the Sequence Listing and a downstream primer P2 having a nucleotide sequence of SEQ. ID. No. 2 of the Sequence Listing.
  • Add product
  • the amplified product was sequenced.
  • Another object of the present invention is to provide a kit for detecting a dystonia VPS16 gene, which comprises a detection primer for the above-mentioned dystonia VPS16 gene in a kit.
  • FIG. 1 is a map of a genetic strain of a dystonia sampled in an embodiment of the present invention
  • Fig. 2 is a graph showing the results of detection of a method for detecting a dystonia VPS16 gene of the present invention.
  • the embodiment of the invention provides a method for detecting a dystonia VPS16 gene, comprising the following steps:
  • the PCR amplification product is sequenced, and the result of the sequencing is compared with the VPS16 gene in the gene bank to verify whether the nucleotide base of the 156th point is mutated from C to A;
  • the primer used in the PCR process of step S20 includes an upstream primer P1 having a nucleotide sequence of SEQ. ID. No. 1 of the Sequence Listing and a downstream primer P2 having a nucleotide sequence of SEQ. ID. No. 2 of the Sequence Listing.
  • the present invention is based on the study of a family of autosomal recessive dystonia, the parents of patients III5 (II4 and II5) are married to close relatives, but the normal performance, the four patients in the world are characterized by early onset, head and neck involuntary movement, The body is twisted, life can not take care of itself, and the initial clinical symptoms are primary dystonia. All living patients were selected as study samples in the family, and II4 and II5 were used as normal sample controls for exon sequencing.
  • the Sanger sequencing method was used to further verify the candidate mutation sites on these two genes. It was found that the c.156C>A homozygous mutation of VPS16 gene was isolated in the family, and the mutation resulted in the coding of the 52th amino acid. .Asn52Lys missense mutation. And 200 normal people do not carry this homozygous gene mutation, so this homozygous gene mutation causes disease.
  • VPS16 c.156C>A heterozygous and homozygous mutant mice as well as VPS16 heterozygous and homozygous knockout mice, were constructed by comparison.
  • the time of movement of the runner fatigue was significantly shortened compared with the normal control mice, and the limbs activity was abnormal by the gait analysis system. Therefore, it was finally determined that VPS16 c.156C>A was indeed a disease-associated gene.
  • the primers for specific design are used in the present invention to amplify the VPS16 gene of the DNA sample to be tested, and then the mutation site is identified.
  • DNA can be extracted from the peripheral blood samples from the collected peripheral blood samples in the peripheral blood samples using the OMEGA Blood DNA Midi Kit Whole Blood DNA Extraction Kit.
  • the genomic DNA is extracted and the DNA content is measured by a spectrophotometer to determine the concentration and purity; and in step S20, the DNA standard with the best concentration and purity is selected for PCR amplification, of course, the primer includes the sequence table.
  • the VPS16 gene amplified by the specific primer in step S20 is sequenced.
  • the amplification product of step S20 can also be purified by a more preferred step prior to sequencing, and the purification can be carried out by gel electrophoresis and/or millipore purification plates.
  • the purification step can remove impurities such as RNA and salt ions, and improve the purity of the amplified fragment.
  • purification of the sequenced amplification product is not necessary to achieve the present invention, purification of the sequenced amplification product can facilitate the accuracy of the sequencing reaction.
  • the results of the sequencing were compared with the VPS16 gene sequence in the normal standard gene bank to see if the base at position 156 was c-mutated to A.
  • the primer pair DNA is used.
  • Direct amplification of the VPS16 gene for analysis and judgment overcomes the one-sidedness of the existing TOR1A, THAP1, GCH1 mutation detection, and the process can be directly realized by ordinary PCR method, which is fast, accurate, efficient, simple, and has a high early diagnosis rate.
  • the test results can provide scientific assistance for the early diagnosis, differential diagnosis and development of dystonia drugs for the treatment of dystonia.
  • the present invention further provides a primer for detecting a dystonia VPS16 gene, and a kit.
  • the primer to be detected therein is of course the upstream primer P1 having the nucleotide sequence of SEQ. ID. No. 1 of the Sequence Listing and the downstream primer P2 having the nucleotide sequence of SEQ. ID. No. 2 of the Sequence Listing.
  • the kit may further comprise a PCR reaction buffer such as 2 ⁇ GC Buffer, an enzyme solution such as LA Taq enzyme, and a positive control of a standard normal VPS16 gene sequencing peak map and the like.
  • a PCR reaction buffer such as 2 ⁇ GC Buffer
  • an enzyme solution such as LA Taq enzyme
  • step S12 The remaining lysate in step S12 was added to a 15 ml centrifuge tube with a filter column, and centrifuged at 4000 rpm for 5 minutes. Remove the filter column, pour off the filtered liquid, and put it back into the filter column.
  • the filter column is moved to a new 15 ml centrifuge tube, 500 ul 70 ° C Elution Buffer is added, allowed to stand at room temperature for 5 minutes, centrifuged at 4000 rpm for 5 minutes, and the DNA-containing filtrate is collected;
  • the filter column is again moved to a new 15 ml centrifuge tube, 500 ul 70 ° C Elution Buffer is added, allowed to stand at room temperature for 5 minutes, centrifuged at 4000 rpm for 5 minutes, and the DNA-containing filtrate is collected;
  • step S20 the DNA standard obtained in step S19 is subjected to PCR, and the process is carried out by using the following system: of course, the primers in the following PCR process adopt the nucleotide sequence of the sequence listing SEQ.ID.No.1 described in the present invention.

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Abstract

本发明提供了一种肌张力障碍VPS16基因的检测引物，其特征在于，包括具有序列表SEQ.ID.No.1碱基序列的上游引物P1和具有序列表SEQ.ID.No.2碱基序列的下游引物P2。本发明的引物，可以实现对标样DNA直接扩增得出VPS16基因进行分析判断，为肌张力障碍的早期诊断、鉴别诊断及开发肌张力障碍治疗药物提供科学辅助。

Description

肌张力障碍VPS16基因的检测引物、方法和试剂盒 技术领域
本发明属于分子生物工程技术领域，具体涉及一种肌张力障碍VPS16基因的检测引物、方法和试剂盒。
背景技术
肌张力障碍是继特发性震颤、帕金森疾病的第三大常见运动障碍性疾病，其临床表现为由于主动肌和拮抗肌收缩不协调，或过度收缩引起的异常动作和姿势，具有不自主性和持续性的特点。目前针对中国肌张力障碍人群的研究中，已发现的致病基因如TOR1A、THAP1、GCH1等，但仅能解释不足5％的病例；因此通过现有致病基因的检测还不能准确地对疑似患病的患者是否患病进行准确判断。
技术问题
本发明实施例的目的在于克服现有技术的上述不足，提供一种克服现有现有TOR1A、THAP1、GCH1等突变检测的片面性的肌张力障碍VPS16基因的检测引物、方法和试剂盒。
发明内容
为了实现上述发明目的，本发明实施例的技术方案如下：
一种肌张力障碍VPS16基因的检测引物，其特征在于，包括具有序列表SEQ.ID.No.1碱基序列的上游引物P1和具有序列表SEQ.ID.No.2碱基序列的下游引物P2。
本发明的上述引物，可以实现对标样DNA直接扩增得出VPS16基因进行分析判断，克服了现有TOR1A、THAP1、GCH1等突变检测的片面性，并且过程直接通过普通PCR方法即可实现，具有快速、准确、高效、简便、早期诊断率高等优点；检测结果可以为肌张力障碍的早期诊断、鉴别诊断及开发肌张力障碍治疗药物提供科学辅助。
本发明进一步还提出采用上述引物进行检测的方法，包括如下步骤：
获取待测DNA标样；
将待测DNA标样通过如具有序列表SEQ.ID.No.1碱基序列的上游引物P1和具有序列表SEQ.ID.No.2碱基序列的下游引物P2进行PCR扩增，得到扩增产物；
将所述扩增产物进行测序。
本发明的另一目的还在于提出一种肌张力障碍VPS16基因的检测试剂盒，在试剂盒中包含有上述肌张力障碍VPS16基因的检测引物。
有益效果
采用本发明的上述检测方法和检测的试剂盒，通过实现扩增VPS16基因然后进行测序，最终通过与正常标准基因库中的VPS16基因序列进行比对，查看第156位点的碱基是否由C突变为A；从而进行结果判断，具有快速、准确、高效、简便、早期诊断率高等优点。
附图说明
下面将结合附图及实施例对本发明作进一步说明，附图中：
图1为本发明实施例中采样的肌张力障碍遗传家系图谱；
图2为本发明肌张力障碍VPS16基因的检测方法的检测结果峰图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明实施例提供一种肌张力障碍VPS16基因的检测方法，包括如下步骤：
S10，制备待测DNA样本；
S20，以待测DNA样本为模板，通过PCR扩增待测DNA样本的VPS16基因；
S30，将PCR扩增产物进行测序，并将测序的结果与基因库中的VPS16基因进行对比，验证第156位点的核苷酸碱基是否由C突变为A；
其中步骤S20的PCR过程中采用的引物包括具有序列表SEQ.ID.No.1碱基序列的上游引物P1和具有序列表SEQ.ID.No.2碱基序列的下游引物P2。
引物名称 序列 长度 序列号编号
上游引物P1 GAAGACAAATGGAGGCTTTAGG 22bp SEQ.ID.No.1
下游引物P2 ACTGCAGGGGAGAGAGGTTC 20bp SEQ.ID.No.2
本发明通过以一个常染色体隐性遗传的肌张力障碍家系的研究，患者III5的父母(II4和II5)为近亲结婚但表现正常，在世的四位患者均表现为早期发病，头颈不自主运动，躯体扭转，生活不能自理，经初步临床症断为原发性肌张力障碍。在家系中选取所有在世的患者作为研究样本，II4和II5作为正常样本对照，进行外显子测序。
具体测序采用利用NimbleGen SeqCap EZ Exome 64M全外显子捕获Kit结合Hiseq 2000高通量测序平台，平均获得133392 SNPs和10797 Indels。随后通过dbSNP数据库、千人基因组数据库、HapMap数据库等公共数据库的过滤，去掉所有已知的且在数据库中等位基因频率大于0.005的变异。通过比对正常样本，去掉所有已知变异，同义突变以及非编码区的变异，并利用SIFT软件进行SNP功能预测，最终得到2个可能引起致病的基因突变。
进一步运用Sanger测序的方法，对这两个基因上的侯选突变位点进一步验证，发现VPS16基因的c.156C>A纯合突变在家系中共分离，该突变导致编码的第52位氨基酸发生p.Asn52Lys错义突变。而200个正常人并不携带此纯合的基因突变，因此这个纯合的基因突变导致疾病。
最终通过自主构建了VPS16 c.156C>A杂合和纯合突变小鼠，以及VPS16杂合和纯合敲除小鼠进行对比验证。在对比缺失VPS16的小鼠肌张力及活动异常，相对于正常对照小鼠，在转轮疲劳仪运动的时间明显缩短，通过步态分析系统发现其四肢活动均有异常。因此最终确定VPS16 c.156C>A确为疾病发生关联基因。
因此在上述情形下，本发明中采用针对特异性设计的引物，扩增待测DNA样本的VPS16基因，然后对突变位点进行鉴定。
在实施过程中步骤S10中DNA样本的获取可以从采集的外周血，对外周血样本中采用OMEGA Blood DNA Midi Kit全血DNA提取试剂盒从外周血样品中提取DNA。
步骤S10完成后，将提取基因组DNA用分光光度计测DNA含量，测定浓度和纯度；进而在步骤S20中选择提取浓度和纯度较好的DNA标样进行PCR扩增，当然其中引物包括具有序列表SEQ.ID.No.1碱基序列的上游引物P1和具有序列表SEQ.ID.No.2碱基序列的下游引物P2。
最终步骤S30将步骤S20以特异性引物扩增得到的VPS16基因进行测序。当然在测序之前更加优选的步骤还可以将步骤S20的扩增产物进行纯化，纯化的方式可以采用凝胶电泳和/或millipore纯化板进行。纯化步骤可以去除RNA、盐离子等杂质，提高扩增片段纯度。虽然测序扩增产物的纯化不是实现本发明所必须，但纯化测序扩增产物可以有利于测序反应的精确度。并进一步通过测序的结果，与正常标准基因库中的VPS16基因序列进行比对，查看第156位点的碱基是否由C突变为A。
本发明中在发现VPS16 c.156C>A这一关联基因后，通过引物对标样DNA 直接扩增得出VPS16基因进行分析判断，克服了现有TOR1A、THAP1、GCH1等突变检测的片面性，并且过程直接通过普通PCR方法即可实现，具有快速、准确、高效、简便、早期诊断率高等优点；检测结果可以为肌张力障碍的早期诊断、鉴别诊断及开发肌张力障碍治疗药物提供科学辅助。
本发明进一步提出一种肌张力障碍VPS16基因的检测的引物、以及试剂盒。其中检测的引物当然为具有序列表SEQ.ID.No.1碱基序列的上游引物P1和具有序列表SEQ.ID.No.2碱基序列的下游引物P2。
试剂盒中除了可以具有上述引物之外，还可以包含PCR反应缓冲液如2×GC Buffer、酶液如LA Taq酶、以及标准正常VPS16基因测序峰图的阳性对照等等。
为使本发明上述引物、方法和试剂盒使用的细节更容易被技术人员理解和实施，以下通过具体实施案例进行举例说明：
实施例1
将从取家系中正常人父亲IV4、儿女V18、以及患者V5(其中家系图谱如附图1所示)的外周血样进行如下步骤：
S10，提取全基因组DNA：
S11，取2ml外周血样本、150ul OB Protease、2.1ml Buffer BL和20ul RNase A加入EP管中，然后最大速度漩涡1分钟彻底混匀后；于65摄氏度水浴15～20分钟，并在水浴过程中漩涡5次；
S12，再向EP管中加入2.2ml无水乙醇，最大速度漩涡30秒，彻底混匀，进行裂解，得裂解液；
S13，将3.5ml裂解液移入带过滤柱的15ml离心管，4000转离心5分钟，取出过滤柱，倒掉过滤液体，放回过滤柱；
将步骤S12剩余裂解液加入带过滤柱的15ml离心管，4000转离心5分钟， 取出过滤柱，倒掉过滤液体，放回过滤柱。
S14，加入3ml HB Buffer，洗涤过滤柱，4000转离心5分钟，取出过滤柱，倒掉过滤液体，放回过滤柱；
S15，加入3ml DNA Wash Buffer，4000转离心5分钟，取出过滤柱，倒掉过滤液体，放回过滤柱；
S16，再次加入3ml DNA Wash Buffer，4000转离心5分钟，取出过滤柱，倒掉过滤液体，放回过滤柱；4000转离心15分钟，甩干过滤柱；
S17，将过滤柱移至新的15ml离心管，加入500ul 70摄氏度的Elution Buffer，室温静置5分钟，4000转离心5分钟，收集含有DNA的过滤液；
S18，再次将过滤柱移至新的15ml离心管，加入500ul 70摄氏度的Elution Buffer，室温静置5分钟，4000转离心5分钟，收集含有DNA的过滤液；
S19，利用分光光度计测量DNA的浓度及纯度，所得的每个标本基因组DNA的OD260/OD280均位于1.7-2.0之间，浓度不少于200ng/ul，总量不少于30μg。
然后步骤S20，将步骤S19所得到的DNA标样进行PCR，过程采用如下体系进行：当然下述PCR过程中引物采用本发明中所描述的具有序列表SEQ.ID.No.1碱基序列的上游引物P1和具有序列表SEQ.ID.No.2碱基序列的下游引物P2；
Figure PCTCN2015075017-appb-000001
95℃预变性变性5min；
然后进行变性-退火-延伸循环：
Figure PCTCN2015075017-appb-000002
Figure PCTCN2015075017-appb-000003
72℃10分钟，最后于4℃中保持备用。
S30，将上述扩增的产物进行测序，所得测序的峰图如图2所示。
从与标准对照的结果中可以看出，父亲IV4、儿女V18的156位点的碱基为C，而患者V5发生156C>A纯合突变。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包括在本发明的保护范围之内。

Claims (8)

  1. 一种肌张力障碍VPS16基因的检测引物，其特征在于，包括具有序列表SEQ.ID.No.1碱基序列的上游引物P1和具有序列表SEQ.ID.No.2碱基序列的下游引物P2。
  2. 一种肌张力障碍VPS16基因的检测方法，其特征在于，包括如下步骤：
    获取待测DNA标样；
    将待测DNA标样通过如权利要求1所述的肌张力障碍VPS16基因的检测引物进行PCR扩增，得到扩增产物；
    将所述扩增产物进行测序。
  3. 如权利要求2所述的肌张力障碍VPS16基因的检测方法，其特征在于，在将所述扩增产物进行测序步骤之后还包括将测序结果与正常标准基因库中的VPS16基因序列进行比对的步骤。
  4. 如权利要求2或3所述的肌张力障碍VPS16基因的检测方法，其特征在于，在将所述扩增产物进行测序步骤之前还包括将所述扩增产物进行纯化的步骤。
  5. 如权利要求4所述的肌张力障碍VPS16基因的检测方法，其特征在于，所述纯化采用凝胶电泳和/或millipore纯化板进行。
  6. 一种肌张力障碍VPS16基因的检测试剂盒，其特征在于，包括权利要求1所述的肌张力障碍VPS16基因的检测引物。
  7. 如权利要求6所述的肌张力障碍VPS16基因的检测试剂盒，其特征在 于，还包括正常VPS16基因测序峰图的阳性对照。
  8. 如权利要求6或7所述的肌张力障碍VPS16基因的检测试剂盒，其特征在于，还包括PCR反应缓冲液和/或DNA聚合酶。
PCT/CN2015/075017 2014-11-03 2015-03-25 肌张力障碍vps16基因的检测引物、方法和试剂盒 WO2016070550A1 (zh)

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