WO2023097884A1 - 一种用于检测jph3基因的引物对、试剂盒及检测方法 - Google Patents

一种用于检测jph3基因的引物对、试剂盒及检测方法 Download PDF

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WO2023097884A1
WO2023097884A1 PCT/CN2022/074505 CN2022074505W WO2023097884A1 WO 2023097884 A1 WO2023097884 A1 WO 2023097884A1 CN 2022074505 W CN2022074505 W CN 2022074505W WO 2023097884 A1 WO2023097884 A1 WO 2023097884A1
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jph3
gene
primer pair
detecting
ctg repeat
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French (fr)
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伍建
姬晓雯
武璇
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迈基诺（重庆）基因科技有限责任公司
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the invention relates to the field of molecular biology, in particular to a primer pair, a kit and a detection method for detecting JPH3 gene.
  • Huntington disease-like 2 is a neurodegenerative disease similar to Huntington disease (HD), and its clinical phenotype, genetic characteristics, neuropathology and development are similar to HD.
  • Huntington's disease-like 2 typically presents in middle age with a persistent triad of motor, mood, and cognitive abnormalities.
  • Neurologic abnormalities including chorea, hypokinesia (rigidity, bradykinesia), dysarthria, and late hyperreflexia have a strong correlation between the duration of the disease and the progression of motor and cognitive impairment, resulting in 10 to Die within 20 years.
  • CTG will have approximately >40 three-base CTG repeat expansion.
  • there is currently no suitable analysis method for the CTG repeat status in the IPH3 gene so it is necessary to design a new detection method to analyze the CTG repeat status in order to analyze the IPH3 gene.
  • the problem to be solved in the present invention is: how to provide a kind of primer pair, kit and method for detecting JPH3 gene, to solve the lack of CTG repeat status analysis method in IPH3 gene in the prior art The problem.
  • the present invention adopts the following technical solutions:
  • the CTG in the HDL2-related IPH3 gene will have approximately more than 40 three-base CTG repeat expansions. Therefore, we can try to design a primer and method for detecting CTG repeat status, and determine the IPH3 gene by determining the number of repeats.
  • the main content of the present invention synthesize a primer pair, at least one of the 5' end of the forward primer and the 5' end of the reverse primer of the primer pair is labeled with a fluorescent group; extract genomic DNA from the sample to be tested as a DNA template; Configure a PCR amplification system comprising the primer pair and the amplification template; perform an amplification reaction on the PCR amplification system to obtain a PCR product containing a CTG trinucleotide repeat sequence; detect the PCR product by capillary electrophoresis , and calculate the JPH3 gene CTG trinucleotide repeat number of the test sample according to the electrophoresis detection result.
  • the present invention provides a pair of primers for detecting dynamic mutations in the CTG repeat sequence of JPH3 gene, at least one of the 5' end of the forward primer and the 5' end of the reverse primer of the primer pair is composed of a fluorescent group Marking, the primer pair sequence is as follows:
  • JPH3-famF GGCAGAGCCGGGGCCGG;
  • JPH3-R GGTTCCCTGCACAGAAACCATC.
  • the primer pair famF carries a 5'-FAM fluorescent group towards the 5' end of the primer.
  • the present invention provides a kit, which includes the above-mentioned primer pair.
  • the kit includes 25 parts by volume of 2xGoldStar Best MasterMix, 2.4 parts by volume of DMSO, 13.6 parts by volume of nuclease-free purified water and 4 parts by volume of the above-mentioned primer pair.
  • the present invention provides a non-diagnostic detection method for detecting the dynamic mutation of the CTG repeat sequence of the JPH3 gene, including:
  • step 1) PCR amplification includes:
  • the amount of 2 ⁇ GoldStar Best Master Mix reagent is 25 ⁇ n ⁇ l
  • the amount of 10uM JPH3-famF primer is 2 ⁇ n ⁇ l
  • the amount of R primers is 2 ⁇ n ⁇ l
  • the amount of DMSO is 2.4 ⁇ n ⁇ l
  • the amount of ddH 2 O is 13.6 ⁇ n ⁇ l
  • the n the number of detection samples+1; add 5ul DNA to the 45ul amplification reaction mixture; carry out PCR amplification.
  • the PCR amplification includes:
  • step 2) performing capillary electrophoresis on the PCR-amplified product of the step 1) includes:
  • the machine mixture was detected in the ABI3730xl sequencer, using G5 color grouping, and the typing parameters in the ABI3730xl sequencer were set as follows: detection constant temperature OVEN_Temperature 60°C, buffer temperature Buffer_Temperature 35°C; pre-electrophoresis voltage PreRun_Voltage 15kV, pre-electrophoresis Time PreRun_Time 180s; injection voltage Injection_Voltage is 1.5kV; injection time Injection_Time is 15s; first reading First_ReadOut_Time 300ms; second reading Second_ReadOut_Time 300ms; electrophoresis voltage Run_Voltage 15kV; val Set to 20s; voltage tolerance Voltage_Tolerance is set to 0.6kV; current Current_Stability is 30uA, temperature change delay Ramp_Delay 1s; date delay Data_Delay 600s; electrophoresis time Run_Time1600s.
  • step 3) analyzing the CTG repeat situation in the JPH3 gene includes:
  • the genemapper ID v3.2 software was used to interpret the typing results. After checking and correcting the internal standard peak, the 61bp point was defined as CTG1.
  • the CTG repeat number corresponding to the peak is the first allele; if there is a fluorescent string peak after that, the CTG repeat number corresponding to the highest peak among the five finger peaks is selected as the second allele.
  • the present invention provides the application of the primer pair for detecting dynamic mutation of CTG repeat sequence of JPH3 gene or the kit for detecting dynamic mutation of CTG repeat sequence of JPH3 gene in the preparation of Huntington's disease-like 2 diagnostic agent.
  • the beneficial effect of the present invention is that: the primer pair, kit and detection method provided by the present invention can evaluate HDL2 by detecting the repeat number of JPH3 gene (CTG) n, and can quickly detect the repetition of JPH3 gene of Huntington's disease syndrome type 2
  • CCG repeat number of JPH3 gene
  • the detection and evaluation are carried out according to the number, the detection process is short and the detection efficiency is high.
  • Figure 1 is a waveform diagram of a sample analyzed by genemapper ID v3.2 in an embodiment of the present invention.
  • Fig. 2 is a waveform diagram of a mock-positive plasmid sample analyzed by genemapper ID v3.2 in the embodiment of the present invention.
  • the present invention designs a pair of specific primers for the CTG repeat of the JPH3 gene. Using this pair of primers to perform PCR amplification on the sample will produce a series of PCR products with different fragment lengths. One end of the product contains fam fluorescence. Capillary electrophoresis is performed on the PCR product, and the number of CTG repeats in the product can be clearly seen. The error is not more than 1 repetition.
  • a pair of specific primers were designed, at least one of the 5' end of the forward primer and the 5' end of the reverse primer of the primer pair was labeled with a fluorophore, and the sequence of the primer pair was as follows:
  • JPH3-famF GGCAGAGCCGGGGCCGG;
  • JPH3-R GGTTCCCTGCACAGAAACCATC.
  • the FAM fluorescent group is selected, and the primer pair famF carries a 5'-FAM fluorescent group to the 5' end of the primer.
  • the sequence of the primer pair is as follows:
  • JPH3-famF GGCAGAGCCGGGGCCGG;
  • JPH3-R GGTTCCCTGCACAGAAACCATC.
  • a pair of primers designed in Example 1 was used to detect blood samples.
  • Example 3 Carry out PCR amplification to the extracted DNA
  • a commercially available PCR kit was used for PCR.
  • the brand of the kit used in this example is Kangwei Century, the name is 2xGoldStar Best MasterMix, and the article number is CW0656S.
  • Reagent configuration configure the amplification reaction liquid mixture according to the number of DNA detection samples, and pack in 45 ul each.
  • the amount of reagents in the amplification reaction liquid mixture is as follows:
  • n the number of detection samples+1.
  • Amplification Carry out the PCR amplification program in the PCR instrument, and the amplification program is as follows:
  • Embodiment 4 Carry out capillary electrophoresis to PCR product
  • Sample dilution The PCR product was diluted 100 times, and 1ul of the PCR amplification product was added to 99ulddH 2 O.
  • Pre-denaturation Run the prepared mixture on the PCR instrument at 95°C for 3 minutes, then cool it in the ice-water mixture for 5 minutes at a high speed for denaturation.
  • Embodiment 5 data analysis
  • the typing results are interpreted in genemapper ID v3.2, and the 61bp position is defined as (CTG)1 after checking and correcting the internal standard peak, and judged according to the number of fluorescent cluster peaks that appear every 3bp or so CTG repeat number, the CTG repeat number corresponding to the first highest peak after (CTG)1 is the first allele, if there are fluorescent string peaks after that, select the CTG repeat number corresponding to the highest peak among the five finger peaks As the second allele, see Figure 1, the CTG repeat number in this sample is 14/16; see Figure 2, the CTG repeat number of the mock-positive plasmid is 40/40, which is consistent with the plasmid synthesis sequence.

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Abstract

涉及分子生物学领域，具体涉及一种用于检测JPH3基因的引物对、试剂盒及检测方法；引物对的正向引物的5'端和反向引物的5'端中的至少一端由荧光基团标记，引物对序列：JPH3-famF：GGCAGAGCCGGGGCCGG；JPH3-R：GGTTCCCTGCACAGAAACCATC；所述方法通过提取DNA并利用上述引物对进行PCR扩增，对PCR扩增后的产物进行毛细管电泳，分析JPH3基因CTG重复序列情况，得出JPH3基因CTG重复数，对HDL2进行评估，整个检测过程耗时短、效率高。

Description

一种用于检测JPH3基因的引物对、试剂盒及检测方法 技术领域
本发明涉及分子生物学领域，具体涉及一种用于检测JPH3基因的引物对、试剂盒及检测方法。
背景技术
亨廷顿病样2(Huntington Disease-like 2,HDL2)是一种类似于亨廷顿病(Huntington Disease,HD)的神经退行性疾病，其临床表型、遗传特征、神经病理学和发展均与HD相似。亨廷顿病样2(HDL2)通常出现在中年，伴有持续的运动、情绪和认知异常三联征。神经系统异常包括舞蹈病、运动减退(僵直、运动迟缓)、构音障碍和后期反射亢进，在疾病的持续时间和运动和认知障碍的进展之间有很强的相关性，会导致10至20年内死亡。
在HDL2相关的IPH3基因中CTG会出现大约大于40个三碱基CTG重复扩增。但目前对IPH3基因中CTG重复状况并没有适配分析方法，所以需要设计新的检测方法对CTG重复状况进行分析，以便对IPH3基因进行分析。
发明内容
针对上述现有技术的不足，本发明所要解决的问题是：如何提供一种用于检测JPH3基因的引物对、试剂盒及方法，以解决现有技术中对IPH3基因中CTG重复状况分析方法缺失的问题。
为了解决上述问题，本发明采用了如下的技术方案：
HDL2相关的IPH3基因中CTG会出现大约大于40个三碱基CTG重复扩增。所以可以尝试设计一种检测CTG重复状况的引物及方法，通过对重复数的确定，以便确定IPH3基因。
本发明主要内容：合成引物对，引物对的正向引物的5’端和反向引物的5’端中的至少一端由荧光基团标记；从待测样本中提取基因组DNA，作为DNA模板；配置包含所述引物对和所述扩增模板的PCR扩增体系；对所述PCR扩增体系进行扩增反应，获得含有CTG三核苷酸重复序列的PCR产物；毛细管电泳检测所述PCR产物，并根据电泳检测结果计算所述待测样本的JPH3基因CTG三核苷酸重 复数。
其一，本发明提供一种用于检测JPH3基因CTG重复序列动态突变的引物对，所述引物对的正向引物的5’端和反向引物的5’端中的至少一端由荧光基团标记，所述引物对序列如下：
JPH3-famF：GGCAGAGCCGGGGCCGG；
JPH3-R：GGTTCCCTGCACAGAAACCATC。
更优的，所述引物对famF向引物5’端带5’-FAM荧光基团。
其二，本发明提供一种试剂盒，所述试剂盒包含上述的引物对。
具体的，所述试剂盒包括25体积份的2xGoldStar Best MasterMix、2.4体积份的DMSO、13.6体积份的无核酸酶纯化水和4体积份上述的引物对。
其三，本发明提供一种检测JPH3基因CTG重复序列动态突变的非诊断目的的检测方法，包括：
1)提取DNA并利用权利要求1所述的引物对进行PCR扩增；
2)对所述步骤1)PCR扩增后的产物进行毛细管电泳；
3)分析JPH3基因中CTG重复情况。
进一步，所述步骤1)PCR扩增包括：
按DNA检测样本数量配置扩增反应液混合液，所述扩增反应液混合液中：2×GoldStar Best Master Mix试剂用量25×nμl；10uM的JPH3-famF引物用量2×nμl；10uM的JPH3-R引物用量2×nμl；DMSO用量2.4×nμl；ddH 2O用量13.6×nμl；所述n＝检测样本数+1；按45ul扩增反应混合液中加入5ul DNA；进行PCR扩增。
具体的，所述PCR扩增包括：
在PCR仪中先以95℃变性1分钟；再运行35次如下循环：94℃变性30秒、59℃退火30秒、72℃延伸1分钟；之后72℃延伸5分钟，4℃保存。
进一步，所述步骤2)对所述步骤1)PCR扩增后的产物进行毛细管电泳包括：
对PCR扩增产物进行稀释，在ddH 2O中加入PCR扩增产物；
取ABI GS500-LIZ内标和HIDI按1∶250的体积比混合后，取混合液与稀释后的PCR扩增产物混合制成上机混合液；
将配置好的上机混合液在PCR仪上以95℃运行3分钟后，冰水混合物中冷却5分钟，确保温度在0℃，作变性处理；
上机混合液在ABI3730xl测序仪中进行检测，采用G5颜色分组，在ABI3730xl测序仪中的分型参数设置如下：检测恒温OVEN_Temperature 60℃，缓冲液温度Buffer_Temperature 35℃；预电泳电压PreRun_Voltage 15kV，预电泳时间PreRun_Time 180s；注入电压Injection_Voltage为1.5kV；注入时间Injection_Time为15s；第一次读数First_ReadOut_Time 300ms；第二次读数Second_ReadOut_Time 300ms；电泳电压Run_Voltage 15kV；突跳步数Voltage_Number_of_steps设置为10步；突跳电压Voltage_steps_Interval设置为20s；电压容差Voltage_Tolerance设置为0.6kV；电流Current_Stability为30uA，变温延迟Ramp_Delay 1s；日期延迟Data_Delay 600s；电泳时间Run_Time1600s。
进一步，所述步骤3)分析JPH3基因中CTG重复情况包括：
采用genemapper ID v3.2软件对分型结果进行判读，核对校正内标峰后定义61bp处为CTG1处，根据每隔3bp左右出现的荧光串峰个数判断CTG重复数，CTG1之后第一个最高峰所对应的CTG重复数为第一个等位基因；若之后还有荧光串峰，则选取五指峰中的最高峰所对应的CTG重复数作为第二个等位基因。
其四，本发明提供所述用于检测JPH3基因CTG重复序列动态突变的引物对或所述用于检测JPH3基因CTG重复序列动态突变的试剂盒在制备亨廷顿病样2诊断剂中的应用。
本发明的有益效果在于：本发明提供的引物对、试剂盒以及检测方法，可以利用检测JPH3基因(CTG)n重复数对HDL2进行评估，可以快速对类亨廷顿病综合征2型JPH3基因的重复数做出检测、评估，检测过程耗时短，检测效率高。
附图说明
为了使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步的详细描述，其中：
图1为本发明实施例中一个样本采用genemapper ID v3.2分析后的波形图。
图2为本发明实施例中一个模拟阳性的质粒样本采用genemapper ID v3.2分析后的波形图。
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明。
需要说明的是，这些实施例仅用于说明本发明，而不是对本发明的限制，在本发明的构思前提下本方法的简单改进，都属于本发明要求保护的范围。
本发明针对JPH3基因的CTG重复，设计了一对特异性的引物。使用这对引物对样本进行PCR扩增，会产生一系列片断长度不等的PCR产物，产物的一端包含有fam荧光，对PCR产物进行毛细管电泳，可以清楚的看到产物中CTG的重复数，误差不超过1次重复。
实施例1
针对JPH3的CTG重复，设计了一对特异性的引物，引物对的正向引物的5’端和反向引物的5’端中的至少一端由荧光基团标记，引物对序列如下：
JPH3-famF：GGCAGAGCCGGGGCCGG；
JPH3-R：GGTTCCCTGCACAGAAACCATC。
选用FAM荧光基团，引物对famF向引物5’端带5’-FAM荧光基团，引物对序列如下：
JPH3-famF：GGCAGAGCCGGGGCCGG；
JPH3-R：GGTTCCCTGCACAGAAACCATC。
实施例2 血液样本的gDNA提取
采用实施例1中设计的一对引物，对血液样本进行检测。
血液样本提取采用天根生化科技(北京)有限公司生产的磁珠法通用型基因组DNA提取试剂盒(DP705-02)：
1、取250μl血液样品至2ml离心管中，加入20μl Proteinase K溶液和300μl裂解液GHL，振荡混匀，75℃裂解15min，期间颠倒混匀3回，每回3-5次。当样本数目比较大时，可以把裂解液GHL和Proteinase K预先混合，现用现配。
2、加入300μl异丙醇，振荡混匀10sec。
3、加入15μl磁珠悬浮液GH，振荡混匀1min，共静置9min，每3min振荡混匀1min。
4、将离心管放置于磁力架上静置30sec，磁珠完全吸附后，吸去液体。
5、加入900μl缓冲液GDZ，振荡混匀2min。
6、将离心管放置于磁力架上静置30sec，磁珠完全吸附后，吸去液体。
7、加入500μl缓冲液GDZ，振荡混匀2min。
8、将离心管放置于磁力架上静置30sec，磁珠完全吸附后，吸去液体。
9、将离心管从磁力架上取下，加入900μl漂洗液PWD(使用前先检查是否已加入无水乙醇)，振荡混匀2min。
10、将离心管放置于磁力架上静置30sec，磁珠完全吸附后，吸去液体。
11、将离心管从磁力架上取下，加入300μl漂洗液PWD，振荡混匀2min。
12、将离心管放置于磁力架上静置30sec，磁珠完全吸附后，吸去液体。
13、将离心管于磁力架上，室温晾干10-15min。乙醇残留会抑制后续的酶反应，所以晾干时要确保乙醇挥发干净。
14、将离心管从磁力架上取下，加入50μl洗脱缓冲液TB，振荡混匀，置于56℃，孵育10min，期间颠倒混匀3回，每回3-5次。
15、将gDNA溶液转移至一个新离心管中，用nanodrop测定其浓度，并根据测定浓度将gDNA浓度浓缩或者稀释至20ng/μL，然后进行后续实验，提取完的gDNA建议立即进行PCR扩增，或者直接放于-20±5℃条件下保存，保存时间不超过一个月。
实施例3 对提取的DNA进行PCR扩增
使用上述引物对对血液样本和模拟阳性的质粒样本(20ng/μL)进行PCR扩增，会产生一系列片断长度不等的PCR产物，产物的一端包含有fam荧光；
PCR采用市售的PCR试剂盒，本实施例采用的试剂盒品牌为康为世纪，名称为2xGoldStar Best MasterMix，货号为CW0656S。
1、试剂配置：按DNA检测样本数量配置扩增反应液混合液，每份45ul分装，所述扩增反应液混合液中试剂用量如下：
成分 用量
2xGoldStar Best MasterMix 25ul×n
HDL2引物Mix 4ul×n
DMSO 2.4ul×n
无核酸酶纯化水 13.6ul×n
总体积 45ul×n
其中，n＝检测样本数+1。
2、加样：在19ul扩增反应混合液中加入1ul DNA；
3、扩增：在PCR仪中进行PCR扩增程序，扩增程序如下：
在PCR仪中先以95℃变性1分钟；再运行35次如下循环：94℃变性30秒、59℃退火30秒、72℃延伸1分钟；之后72℃延伸5分钟，4℃保存。
实施例4 对PCR产物进行毛细管电泳
1、样本稀释：对PCR产物进行100倍样本稀释，在99ulddH 2O中加入1ulPCR扩增产物。
2、配置上机混合液：取ABI GS500-LIZ内标和HIDI按1∶250体积比混合后，取9ul混合液与1ul稀释后的PCR扩增产物混合制成上机混合液。
3、预变性：将配置好的上机混合液在PCR仪上95℃运行3分钟后，冰水混合物中极速冷却5分钟，作变性处理。
4、毛细管电泳：上机混合液在ABI3730xl测序仪中进行检测，采用G5颜色分组，在ABI3730xl测序仪中分型参数设置如下表所示。
Name Value
OVEN_Temperature 60℃
Buffer_Temperature 35℃
PreRun_Voltage 15kV
PreRun_Time 180s
Injection_Voltage 1.5kV
Injection_Time 15s
First_ReadOut_Time 300ms
Second_ReadOut_Time 300ms
Run_Voltage 15kV
Voltage_Number_of_steps 10
Voltage_steps_Interval 20s
Voltage_Tolerance 0.6kV
Current_Stability 30uA
Ramp_Delay 1s
Data_Delay 600s
Run_Time 1600s
实施例5 数据分析
3730xl测序仪分型完成后在genemapper ID v3.2中对分型结果进行判读，核对校正内标峰后定义61bp处为(CTG)1处，根据每隔3bp左右出现的荧光串峰个数判断CTG重复数，(CTG)1之后第一个最高峰所对应的CTG重复数为第一个等位基因，之后如果还有荧光串峰，则选取五指峰中的最高峰所对应的CTG重复数作为第二个等位基因，参见图1，该样本中CTG重复数为14/16；参见图2，该模拟阳性的质粒CTG重复数为40/40，与质粒合成序列一致。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管通过参照本发明的优选实施例已经对本发明进行了描述，但本领域的普通技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离所附权利要求书所限定的本发明的精神和范围。

Claims (10)

  1. 一种用于检测JPH3基因CTG重复序列动态突变的引物对，其特征在于，所述引物对的正向引物的5’端和反向引物的5’端中的至少一端由荧光基团标记，所述引物对序列如下：
    JPH3-famF：GGCAGAGCCGGGGCCGG；
    JPH3-R：GGTTCCCTGCACAGAAACCATC。
  2. 根据权利要求1所述用于检测JPH3基因CTG重复序列动态突变的引物对，其特征在于，所述引物对famF向引物5’端带5’-FAM荧光基团。
  3. 一种用于检测JPH3基因CTG重复序列动态突变的试剂盒，其特征在于，所述试剂盒包含权利要求1所述的引物对。
  4. 根据权利要求3所述用于检测JPH3基因CTG重复序列动态突变的试剂盒，其特征在于，所述试剂盒包括25体积份的2xGoldStar Best MasterMix、2.4体积份的DMSO、13.6体积份的无核酸酶纯化水和4体积份权利要求1所述的引物对。
  5. 一种检测JPH3基因CTG重复序列动态突变的非诊断目的的检测方法，其特征在于，包括：
    1)提取DNA并利用权利要求1所述的引物对进行PCR扩增；
    2)对所述步骤1)PCR扩增后的产物进行毛细管电泳；
    3)分析JPH3基因中CTG重复情况。
  6. 根据权利要求5所述检测JPH3基因CTG重复序列动态突变的非诊断目的的检测方法，其特征在于，所述步骤1)PCR扩增包括：
    按DNA检测样本数量配置扩增反应液混合液，所述扩增反应液混合液中：2×GoldStar Best Master Mix试剂用量25×nμl；10uM的JPH3-famF引物用量2×nμl；10uM的JPH3-R引物用量2×nμl；DMSO用量2.4×nμl；ddH 2O用量13.6×nμl；所述n＝检测样本数+1；按45ul扩增反应混合液中加入5ulDNA；进行PCR扩增。
  7. 根据权利要求6所述检测JPH3基因CTG重复序列动态突变的非诊断目的的检测方法，其特征在于，所述PCR扩增包括：
    在PCR仪中先以95℃变性1分钟；再运行35次如下循环：94℃变性30秒、59℃退火30秒、72℃延伸1分钟；之后72℃延伸5分钟，4℃保存。
  8. 根据权利要求5所述检测JPH3基因CTG重复序列动态突变的非诊断目的的检测方法，其特征在于，所述步骤2)对所述步骤1)PCR扩增后的产物进行毛细管电泳包括：
    对PCR扩增产物进行稀释，在ddH 2O中加入PCR扩增产物；取ABI GS500-LIZ内标和HIDI按1∶250的体积比混合后，取混合液与稀释后的PCR扩增产物混合制成上机混合液；将配置好的上机混合液在PCR仪上以95℃运行3分钟后，冰水混合物中冷却5分钟，温度为0℃，作变性处理；
    上机混合液在ABI3730xl测序仪中进行检测，采用G5颜色分组，在ABI3730xl测序仪中的分型参数设置如下：检测恒温OVEN_Temperature 60℃，缓冲液温度Buffer_Temperature 35℃；预电泳电压PreRun_Voltage 15kV，预电泳时间PreRun_Time 180s；注入电压Injection_Voltage为1.5kV；注入时间Injection_Time为15s；第一次读数First_ReadOut_Time 300ms；第二次读数Second_ReadOut_Time 300ms；电泳电压Run_Voltage 15kV；突跳步数Voltage_Number_of_steps设置为10步；突跳电压Voltage_steps_Interval设置为20s；电压容差Voltage_Tolerance设置为0.6kV；电流Current_Stability为30uA，变温延迟Ramp_Delay 1s；日期延迟Data_Delay 600s；电泳时间Run_Time 1600s。
  9. 根据权利要求5所述检测JPH3基因CTG重复序列动态突变的非诊断目的的检测方法，其特征在于，所述步骤3)分析JPH3基因中CTG重复情况包括：
    采用genemapper ID v3.2软件对分型结果进行判读，核对校正内标峰后定义61bp处为CTG1处，根据每隔3bp左右出现的荧光串峰个数判断CTG重复数，CTG1之后第一个最高峰所对应的CTG重复数为第一个等位基因；若之后还有荧光串峰，则选取五指峰中的最高峰所对应的CTG重复数作为第二个等位基因。
  10. 权利要求1所述用于检测JPH3基因CTG重复序列动态突变的引物对或权利要求3所述用于检测JPH3基因CTG重复序列动态突变的试剂盒在制备亨廷顿病样2诊断剂中的应用。
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