CN112522385A - 一种检测dnm2基因拷贝数变异的试剂盒 - Google Patents
一种检测dnm2基因拷贝数变异的试剂盒 Download PDFInfo
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Abstract
本发明公开了一种检测DNM2基因拷贝数变异的试剂盒。所述试剂盒中含有如下引物对:Q‑DNM2‑LF:GGAGCAACGGCTACAGACCGC;Q‑DNM2‑LR:CTCTTCCATCCCGCGGTTGGGC;Q‑DNM2‑MF:GGACGCCTTCAGCTCCATGCGC;Q‑DNM2‑MR:CGGCCCACGAAGTTCTCCAGCTGC;Q‑DNM2‑RF:CCAAGGTCTTCTTGGGCACGG;Q‑DNM2‑RR:CCACTGGGCCAGGCTTGCCGCACG。本发明简单快捷,预实验成本低,适用于小批量检测和验证。
Description
技术领域
本发明属于生物医学检测技术领域,具体涉及一种检测DNM2基因拷贝数变异的试剂盒。
背景技术
DNM2(dynamin 2)基因是一种转录因子,参与多种细胞功能,如胞吞,吞噬体形成,细胞内运输,与肌动蛋白和微管网相互作用,以及促进细胞凋亡等。DNM2基因如发生致病变异可引起中央核性肌病(CNM)1型和腓骨肌萎缩症,以及致命性先天性挛缩综合征。研究发现CNM与DNM2基因突变本身关系并不明显,而与其mRNA表达水平密切相关,降低其表达水平可明显改善病变,该现象提示CNM发病存在独特分子机制。前期研究发现DNM2含有多个intragenic miRNAs,肌肉损伤时intragenic miR-199a-1常与DNM2表达同步升高,而干预miR-199a-1的水平可改变肌肉的修复进程和中央核肌纤维比例。由此提出:CNM可能是由DNM2的intragenic miRNA表达过高引起的,该miRNA通过调控靶基因表达延迟肌肉分化,导致中央核肌纤维比例增多。
拷贝数变异(Copy number variation,CNV)一般是指长度为1kb以上的基因组大片段的拷贝数增加或者减少,主要表现为亚显微水平的缺失、重复和倒位。与SNP相比,CNV数量虽然不多,但涉及更多的基因组序列,突变率更高。CNV是人类遗传多样性和个体间遗传差异的一个重要原因,除了一部分会致病以外也可以作为遗传多态性的一种。2004年,两项里程碑式的研究发现这种DNA拷贝数亚微观的变异(<500kb)广泛存在于正常人类基因组中。
CNV是基因组结构变异(Structural variation,SV)的重要组成部分,过去几年中,随着基因组信息的不断挖掘以及检测技术的进步,CNV在遗传病发病中的作用越来越受到重视,其致病机制可能与基因剂量效应、基因断裂、基因融合和位置效应等有关。对CNV的深入研究,可能有助于更好的理解遗传性疾病的发病机制,有利于分子诊断和治疗手段的研究和开发。
基于PCR技术的方法中应用最为广泛的是qPCR,它利用相对定量实时PCR***,对检测样本的目标基因(具有拷贝数多态)以及参照基因(无拷贝数多态),利用2^-ΔΔCt相对定量分析法(Livak and Schmittgen,2001)进行拷贝数的分析。该方法简单易于操作,敏感性高,重复性好,速度快和污染少,但是不适合大样本的高通量检测。
发明内容
本发明旨在克服现有技术中的不足,提供一种检测DNM2基因拷贝数变异的试剂盒。
为了达到上述目的,本发明提供的技术方案为:
所述检测DNM2基因拷贝数变异的试剂盒中含有如下引物对:
Q-DNM2-LF:GGAGCAACGGCTACAGACCGC(SEQ ID NO.1)
Q-DNM2-LR:CTCTTCCATCCCGCGGTTGGGC(SEQ ID NO.2)
Q-DNM2-MF:GGACGCCTTCAGCTCCATGCGC(SEQ ID NO.3)
Q-DNM2-MR:CGGCCCACGAAGTTCTCCAGCTGC(SEQ ID NO.4)
Q-DNM2-RF:CCAAGGTCTTCTTGGGCACGG(SEQ ID NO.5)
Q-DNM2-RR:CCACTGGGCCAGGCTTGCCGCACG(SEQ ID NO.6)。
优选地,所述试剂盒中还含有内参引物对:
Q-RP-F:GCAGATTTGGACCTGCGACGGG(SEQ ID NO.7)
Q-RP-R:GTGAGCGGCTGTCTCCACAACACC(SEQ ID NO.8)。
使用上述试剂盒中的引物对进行扩增的程序如下:
①:95℃,5min;
②:95℃,10s;60℃,30s;40Cycles;
③:95℃,15s;60℃,60s;95℃,15s。
qPCR所使用的荧光化学可分为TaqMan荧光探针和SYBR Green荧光染料两种。本发明中,利用SYBR Green荧光染料法进行拷贝数检测的具体原理为:目标序列为正常(拷贝数=2)序列时,目标序列和内参序列的表达量一致,且预试验中已通过引物优化确保二者的扩增效率一致,所以目标序列的C t和内参序列的C t值相等,表现在扩增曲线上则为目标序列的扩增曲线和内参序列的扩增曲线吻合在一起。根据2^-ΔΔCt公式计算出的归一化比值(normalized ratio,NR)为1左右。目标序列存在单拷贝缺失时(拷贝数=1)时,其表达量相当于内参基因RP的1/2,C t值增加1左右,目标基因的扩增曲线在内参基因RP的扩增曲线后面,经2^-ΔΔCt公式计算的NR值在0.5左右。双拷贝缺失时(拷贝数=0),没有目标基因,其扩增曲线和阴性对照的扩增曲线相似,根据2^-ΔΔCt公式计算的NR值在0左右。目标基因拷贝数增加时(拷贝数>2)时,目标基因相对于内参基因RP表达量增加,C t值降低,目标基因的扩增曲线在内参基因RP的扩增曲线前面且目标基因拷贝数增加越多,其扩增曲线和内参基因RP的扩增曲线分开越远。当单拷贝增加时(拷贝数=3),经2^-ΔΔCt公式计算的NR值在1.5左右;当双拷贝增加时(拷贝数=4),经2^-ΔΔCt公式计算的NR值在2.0左右,其它情况的拷贝数重复以此类推。
引物扩增长度一般在100bp左右,不宜过长或过短;设计好的引物应该避免其退火部位出现发夹结构、单核苷酸多态(SNP)位点;避免引物间形成二聚体,引物扩增效率最好接近100%。本发明中,通过UCSC数据库获得DNM2基因1号外显子(chr19:10828909-10829089;hg19)及前后两端的碱基序列,利用UCSC中tools中的Blat确认待测区域存在的同源序列或假基因区域,选择的引物序列避开同源区域,确认其退火温度,是否存在二级结构,在引物偏5’端位置改变一到两个碱基减少二级结构,如Q-DNM2-RR引物,由CACTGGGCCAGGCTTGCCGCTGG改为CCACTGGGCCAGGCTTGCCGCACG(SEQ ID NO.6)。最终设计(L/M/R)3组短片段引物对,另增一对内参引物RP(RPP30),特异性强,检测精准。
本发明通过QPCR的方法,利用Syber Green荧光染料法检测DNM2基因1号外显子上发生拷贝数变异(染色体绝对位置为:chr19:10828909-10829089;hg19)。相对于二代测序成本高、拷贝数变化检测结果准确性不足等问题,本发明简单快捷,预实验成本低,适用于小批量检测和验证。
附图说明
图1:Q-DNM2-LF+Q-DNM2-LR优化前扩增效率;
图2:Q-DNM2-MF+Q-DNM2-MR优化前扩增效率;
图3:Q-DNM2-RF+Q-DNM2-RR优化前扩增效率;
图4:Q-RP-F+Q-RP-R优化前扩增效率;
图5:Q-DNM2-LF+Q-DNM2-LR优化后扩增效率;
图6:Q-DNM2-MF+Q-DNM2-MR优化后扩增效率;
图7:Q-DNM2-RF+Q-DNM2-RR优化后扩增效率;
图8:Q-RP-F+Q-RP-R优化后扩增效率;
图9:Q-DNM2-LF+Q-DNM2-LR优化前溶解曲线;
图10:Q-DNM2-MF+Q-DNM2-MR优化前溶解曲线;
图11:Q-DNM2-RF+Q-DNM2-RR优化前溶解曲线;
图12:Q-RP-F+Q-RP-R优化前溶解曲线;
图13:DNM2-LF+Q-DNM2-LR优化后溶解曲线;
图14:Q-DNM2-MF+Q-DNM2-MR优化后溶解曲线;
图15:Q-DNM2-RF+Q-DNM2-RR优化后溶解曲线;
图16:Q-RP-F+Q-RP-R优化后溶解曲线。
具体实施方式
1、引物测试
1)线性DNA准备(表1):将标准品Human genetic DNA(253ng/ul)梯度稀释,取13ul加入37ul ddH2O,涡旋,微离,浓度即为66ng/uL。
然后10倍梯度稀释,取20ul加入到180ul ddH2O中,每次均需要充分涡旋混匀后才能进行下个梯度稀释。
表1
2)实验体系(表2)
表2
3)扩增程序(表3)
表3
4)结果分析
ABI7500设置:
Experiment Prop选择Quantutation–Relative Standard Curve and SYBRGreen Reagents.
结果如下:
引物DNM2-L:引物扩增效率92.86%,溶解曲线峰单一;
引物DNM2-M:引物扩增效率95.1%,溶解曲线峰单一;
引物DNM2-R:引物扩增效率93.34%,溶解曲线峰单一;
引物RP:引物扩增效率94.55%,溶解曲线峰单一;
引物扩增效率:PCR手册规定,目的基因和内参基因RP这两对引物的PCR扩增效率差异不超过10%的情况下,可进行相似相对定量计算。为此我们规定E值在90—110%之间的引物为合格。引物优化前后的扩增效率,见图1-8,由图可知,引物优化前,扩增效率不在90%-110%范围内,扩增效率差,而优化后,扩增效率接近100%,扩增效率高。
优化前引物如下:
Q-DNM2-LF:GAGCAACGGCTACAGACGCC(SEQ ID NO.9)
Q-DNM2-LR:CTTCCATCCCGCGGTTGCC(SEQ ID NO.10)
Q-DNM2-MF:GACGCCTTCAGCTCCATCGG(SEQ ID NO.11)
Q-DNM2-MR:GCCCACGAAGTTCTCCAGCA(SEQ ID NO.12)
Q-DNM2-RF:CAAGGTCTTCTTGGGCTGG(SEQ ID NO.13)
Q-DNM2-RR:CACTGGGCCAGGCTTGCCGCTGG(SEQ ID NO.14)。
优化前内参引物为:
Q-RP-F:AGATTTGGACCTGCGAGCG(SEQ ID NO.15)
Q-RP-R:GAGCGGCTGTCTCCACAAGT(SEQ ID NO.16)。
优化后的引物及内参引物即为本发明前述SEQ ID NO.1至SEQ ID NO.8所示的引物。
溶解曲线:因为SYBR Green染料并不能只特异性的结合目的基因,所以必须保证引物的特异性扩增,可以进行熔解曲线分析,出现均一熔解峰为合格。如被淘汰的DNM2.W2R,非特异的扩增会导致拷贝数计算出现较大误差(非单一溶解曲线峰)。引物优化前后的溶解曲线图,见图9-16,由图可知,引物优化前,溶解曲线峰不单一,引物非特异性扩增,效果差,而优化后,溶解曲线峰单一,引物特异性扩增,效果好。
2、临床样本检测
临床检测样本,为二代测序Nextseq 550平台检测样本,结果提示其在chr19:10828909-10829089区域测序深度低于相邻两个外显子区一半,考虑杂合缺失可能,其IGV结果如下:chr19:10828890-10829162区域测序深度小于56,chr19:10822038-10839535区域测序深度约114,chr19:10861715-10879212区域测序深度约93。
结果验证
1)将2×AceQ SYBR qPCR Master Mix等试剂、稀释成5μM引物在常温解冻,准备EP管、枪头、加样枪。
2)试剂、引物轻微震荡后瞬时离心。根据引物种类标记EP管。
3)按照检测项目配制体系(表4):
表4
4)试剂及样本设置
配制好的试剂加入到96孔光学PCR板中,准备加样(图1)。以12个孔为一组,加入同一样本;其中,一部分为目的基因引物体系,另一部分为内参基因RP引物体系,均为三个复孔。在每个引物检测中必须包含一个正常人样本STD和一个NTC。本次实验中的Sample1、2、3分别对应上述二代测序样本及其父母。
5)测定样品DNA浓度,并且稀释至5ng/ul,吸取2ul稀释后的样本到对应孔中。盖好封膜后短时离心,准备上ABI7500扩增。
6)上机ABI7500,软件设置。
(1)Experiment properties:
实验类型:Quantitation-comparative CT(△△CT)
试剂类型:SYBR@Green Regents
其他参数默认。
(2)Plate setup:
Define target:分别命名目的基因和内参基因RP;
Define sample:对样品进行命名;
Assign target and sample:对命名好的基因、样本进行位置勾选;
Reference sample选择为STD;
Endogenous control选择RP;
Select the dye use for passive refenence选择ROX。
其他参数默认。
(3)Run method:选择实验体系为20ul(表5);
表5
7)结果分析
通过导出原始CT数据(表6):
表6
Sample1 | Sample2 | Sample3 | STD | |
DNM2-L | 27.15 | 26.89 | 27.22 | 26.07 |
DNM2-R | 25.80 | 25.66 | 25.89 | 24.88 |
DNM2-M | 26.23 | 26.09 | 26.31 | 25.33 |
RP | 25.90 | 26.48 | 25.96 | 25.67 |
ΔCt=Ct(目的基因)-Ct(内参基因)
ΔΔCt=ΔCt(待测样本)-ΔCt(STD)
拷贝数=2*(2^–ΔΔCt)
根据计算公式计算后各数据的2*(2^–ΔΔCt)值(表7)
表7
Sample1 | Sample2 | Sample3 | |
DNM2-L | 1.11 | 1.97 | 1.10 |
DNM2-R | 1.24 | 2.04 | 1.21 |
DNM2-M | 1.26 | 2.07 | 1.24 |
各样本数据的拷贝数(表8)
表8
Sample1 | Sample2 | Sample3 | |
DNM2-L | 单拷贝 | 双拷贝 | 单拷贝 |
DNM2-R | 单拷贝 | 双拷贝 | 单拷贝 |
DNM2-M | 单拷贝 | 双拷贝 | 单拷贝 |
结果表明,3组引物对于同一样本,计算结果相近,符合预期,所得拷贝数结果与二代测序一致,并示其缺失来自于母亲。所涉及引物可用于DNM2基因1号外显子(chr19:10828909-10829089;hg19)区拷贝数变异检测。
序列表
<110> 长沙金域医学检验实验室有限公司
<120> 一种检测DNM2基因拷贝数变异的试剂盒
<141> 2020-12-11
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> null
<400> 1
ggagcaacgg ctacagaccg c 21
<210> 2
<211> 22
<212> DNA
<213> null
<400> 2
ctcttccatc ccgcggttgg gc 22
<210> 3
<211> 22
<212> DNA
<213> null
<400> 3
ggacgccttc agctccatgc gc 22
<210> 4
<211> 24
<212> DNA
<213> null
<400> 4
cggcccacga agttctccag ctgc 24
<210> 5
<211> 21
<212> DNA
<213> null
<400> 5
ccaaggtctt cttgggcacg g 21
<210> 6
<211> 24
<212> DNA
<213> null
<400> 6
ccactgggcc aggcttgccg cacg 24
<210> 7
<211> 22
<212> DNA
<213> null
<400> 7
gcagatttgg acctgcgacg gg 22
<210> 8
<211> 24
<212> DNA
<213> null
<400> 8
gtgagcggct gtctccacaa cacc 24
<210> 9
<211> 20
<212> DNA
<213> null
<400> 9
gagcaacggc tacagacgcc 20
<210> 10
<211> 19
<212> DNA
<213> null
<400> 10
cttccatccc gcggttgcc 19
<210> 11
<211> 20
<212> DNA
<213> null
<400> 11
gacgccttca gctccatcgg 20
<210> 12
<211> 20
<212> DNA
<213> null
<400> 12
gcccacgaag ttctccagca 20
<210> 13
<211> 19
<212> DNA
<213> null
<400> 13
caaggtcttc ttgggctgg 19
<210> 14
<211> 23
<212> DNA
<213> null
<400> 14
cactgggcca ggcttgccgc tgg 23
<210> 15
<211> 19
<212> DNA
<213> null
<400> 15
agatttggac ctgcgagcg 19
<210> 16
<211> 20
<212> DNA
<213> null
<400> 16
gagcggctgt ctccacaagt 20
Claims (3)
1.一种检测DNM2基因拷贝数变异的试剂盒,其特征在于,所述试剂盒中含有如下引物对:
Q-DNM2-LF:GGAGCAACGGCTACAGACCGC
Q-DNM2-LR:CTCTTCCATCCCGCGGTTGGGC
Q-DNM2-MF:GGACGCCTTCAGCTCCATGCGC
Q-DNM2-MR:CGGCCCACGAAGTTCTCCAGCTGC
Q-DNM2-RF:CCAAGGTCTTCTTGGGCACGG
Q-DNM2-RR:CCACTGGGCCAGGCTTGCCGCACG。
2.如权利要求1所述的试剂盒,其特征在于,所述试剂盒中还含有内参引物对:
Q-RP-F:GCAGATTTGGACCTGCGACGGG
Q-RP-R:GTGAGCGGCTGTCTCCACAACACC。
3.如权利要求2所述的试剂盒,其特征在于,使用所述试剂盒中的引物对进行扩增的程序如下:
①:95℃,5min;
②:95℃,10s;60℃,30s;40Cycles;
③:95℃,15s;60℃,60s;95℃,15s。
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