CN116694753A - 检测先天性血小板缺陷的引物组合物、试剂盒及方法 - Google Patents
检测先天性血小板缺陷的引物组合物、试剂盒及方法 Download PDFInfo
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Abstract
本发明涉及生物技术领域,具体公开了一种检测先天性血小板缺陷的引物组合物、试剂盒及方法,该引物组合物能够检测先天性血小板缺陷基因的1075个突变位点的至少一个,所述的1075个突变位点位于49个基因上。该引物组合物能够检测先天性血小板缺陷基因,具有操作简便,精确度、准确性、灵敏度高优点,解决了目前检测准确性低,检测方法局限的技术缺陷,且该引物组合物可一次性检测所述49个基因的1075个基因突变位点,覆盖面广,检测效率高。
Description
技术领域
本发明属于生物技术领域,特别涉及血小板发育研究领域,具体涉及检测先天性血小板缺陷的引物组合物、试剂盒及方法。
背景技术
先天性血小板缺陷是一类少见的遗传性疾病,具体包括血小板无力症、遗传性血小板减少症、巨大血小板综合症等疾病,主要是由于巨核细胞分化产板、血小板骨架发育、血小板颗粒形成以及血小板信号转导过程中相关基因发生致病突变导致血小板数量减少和/或功能异常,大多数呈常染色体隐性遗传,往往自幼即有出血倾向。
常规的实验室诊断方法仅有形态学或者血常规检测:形态学方法仅能主观上初步判断血小板的体积大小或是巨核细胞的形态异常,但是缺乏特征性指标或者判断标准;血常规中包含的血小板计数、平均血小板体积以及血小板压积等指标亦不能够满足对此类疾病的诊断要求,尤其是在血小板数量较低的情况时,血小板相关量化指标可能存在失真。此外血小板相关功能实验操作繁琐复杂,对于标本采集和质量要求极高,并且仍然存在很大误诊或漏诊的可能。
近年来,对于先天性血小板缺陷分子发病基础研究取得了显著进展,目前已知的主要关于先天性血小板缺陷的致病基因有40余种。
通过基因检测能够快速有效的对疾病进行诊断并作出分型,其中分子生物学的实验方法主要有PCR、一代测序等。杜燕华等(实时荧光PCR法与ELISA法在发热伴血小板减少综合征病例检测中的比较[J],中国人兽共患病学报,2013,29(1):101-104)公开了实时荧光PCR法在发热伴血小板减少综合征病例检测中的应用,对发热伴血小板减少综合征监测病例的急性期血清,分别应用实时荧光PCR法和ELISA-IgM方法进行检测,结果143例病例经实时荧光PCR法检测,阳性率50.35%,经ELISA-IgM法检测,阳性率为37.76%,两种方法的检测有统计学意义(P<0.05)。对于间隔时间超过1w的病例,两种检测方法无统计学意义(P>0.05),数据表明实时荧光PCR法更适合于发热伴血小板减少综合征病例的早期实验室诊断,且PCR方法不能直观的得到具的突变序列;一代测序的方法受到测序通量的影响,无法一次性检测多个基因的全部外显子区域,而二代测序作为一种高通量的检测方法,可以一次性准确检测多个基因的外显子以及相关序列,使精准诊断先天性血小板缺陷成为可能。当前虽然先天性血小板缺陷突变基因及位点众多,但是现已知的突变位点较少,缺少使先天性血小板缺陷基因诊断的更加全面、覆盖面更广的突变位点及其检测试剂盒。
发明内容
除非上下文另有明确指示,否则如本文所用的单数形式“一个”、“一种”和“所述”包括单数和复数指示物。通过端点表述的数值范围包括在对应范围内的所有数值和分数,以及所表述的端点。
本发明针对现有技术存在的问题,提供了一种检测先天性血小板缺陷的引物组合物、试剂盒及方法。包含该引物组合物的试剂盒能够更加全面的诊断先天性血小板缺陷相关基因突变的基因群,结合二代测序技术,操作更方便,精确度、准确性和灵敏度更高。
为实现上述目的,本发明采用的技术方案如下:
一方面,本发明提供了一组检测先天性血小板缺陷基因的突变位点的引物组合物,所述的突变位点位于ACBD5、ACTN1、ANKRD26、ANO6、AP3B1、BLOC1S3、BLOC1S6、CYCS、DTNBP1、ETV6、FERMT3、GATA1、GFI1B、GNAS、GNE、GP1BA、GP1BB、GP6、GP9、HOXA11、HPS1、HPS3、HPS4、HPS5、HPS6、ITGA2B、ITGB3、LYST、MASTL、MPL、MYH9、NBEA、NBEAL2、ORAI1、P2RY12、PLA2G4A、PRKACG、RASGRP2、RBM8A、RUNX1、STIM1、STXBP2、TBXA2R、TBXAS1、THPO、TUBB1、VIPAS39、VPS33B、WAS共49个基因上;所述的引物组合物由表1所示引物组成。
表1检测先天性血小板缺陷基因的突变位点的引物组合物
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具体地,所述引物组合物中的每条引物浓度为100nM。
优选地,所述的引物组合物能够扩增上述的突变位点中的至少一个。具体地,所述的突变位点信息如表2所示:
表2突变位点信息
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本发明还提供了一种上述的引物组合物在制备检测先天性血小板缺陷基因的试剂盒中的应用。
本发明还提供了一种检测先天性血小板缺陷基因的试剂盒,所述的试剂盒包括上述的引物组合物。
优选地,所述的试剂盒还包括将基因组DNA制成可供测序的文库的试剂。
优选地,所述的试剂盒还包括用于二代测序的试剂。
优选地,所述的试剂盒还包括DNA聚合酶、缓冲液、ddH2O、dNTP。
再一方面,本发明提供了一种检测先天性血小板缺陷基因的方法,所述的方法包括利用上述的试剂盒对待测样品中先天性血小板缺陷基因进行检测。
所述的方法为非疾病诊断和治疗方法。
优选地,所述的方法,包括以下步骤:
S1、样品基因组DNA提取;
S2、对步骤S1中的DNA进行PCR扩增;
S3、文库构建;
S4、文库测序及数据处理分析。
相对于现有技术,本发明具有以下有益效果:
(1)利用本发明提供的与先天性血小板缺陷相关基因突变的基因群,结合高通量测序技术,可以实现在骨髓形态学和血常规检查之外对先天性血小板缺陷进行辅助诊断,特别是对于疾病后续的治疗方向和效果做出预判具有特殊的优势,可弥补临床先天性血小板缺陷在分子诊断、疾病演变、治疗、预后、用药指导方面的不足。
(2)本发明中的引物组合物能够检测先天性血小板缺陷基因,具有操作简便,精确度、准确性、灵敏度高优点,解决了目前检测准确性低,检测方法局限的技术缺陷。
(3)本发明中的引物组合物可一次性检测所述49个基因的1075个基因突变位点,覆盖面广,检测效率高。
具体实施方式
下面通过具体实施例对本发明进行说明,以使本发明技术方案更易于理解、掌握,但本发明并不局限于此,描述的实施例仅是本申请一部分实施例,而不是全部的实施例。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例,都应当属于本申请保护的范围。下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著,黄培堂等译的《分子克隆实验指南》,第三版,科学出版社)或者按照产品说明书进行。
实施例1先天性血小板缺陷突变基因信息及测序
1、采用dbSNP数据库(http://www.ncbi.nlm.nih.gov/snp/),千人基因组数据库1000Genomes(http://www.1000genomes.org/),外显子组整合数据库ExAC(http://exac.broadinstitute.org/)和人类基因突变数据库HGMD(http://www.hgmd.cf.ac.uk/ac/index.php),以及功能预测工具PolyPhen(http://genetics.bwh.harvard.edu/pph2/)和SIFT(http://sift.jcvi.org/),并采用筛选原则筛选致病突变位点。
筛选原则:(1)根据突变所在的基因组位置和类型,过滤掉对蛋白产物序列不产生影响的突变;(2)利用1000Genomes数据和ExAC数据,注释每个突变在人群中的比例,如果比例小于等于1%则不认为是多态性位点;(3)检索人类基因突变数据库,查询一个突变在HGMD中是否有记载,其出现在SCN等相关疾病中;(4)利用蛋白质功能预测软件PolyPhen-2和SIFT预测该突变是否影响蛋白功能;(5)如果经过(2)(3)(4)后,一个突变满足“非多态性”、“SCN记载过”和“影响蛋白功能”这三条中至少两条的,将被判为与疾病可能相关;(6)如果一个突变虽然不满足(5)但是在HGMD中有记录,则被判读为意义不明的突变;如果一个突变在文献中被明确记载与疾病高度相关,则直接判为热点突变。
最终筛选得到以下基因:(1)ACBD5基因与遗传性血小板减少症2型相关;(2)ACTN1基因与遗传性巨血小板减少症(血小板型出血性疾病15)相关;(3)ANKRD26基因与遗传性血小板减少症2型相关;(4)ANO6基因与Scott综合症相关;(5)AP3B1基因与Hermansky-Pudlak综合征相关;(6)BLOC1S3基因与Hermansky-Pudlak综合征相关;(7)BLOC1S6基因与Hermansky-Pudlak综合征相关;(8)CYCS基因与遗传性血小板减少症4型相关;(9)DTNBP1基因与Hermansky-Pudlak综合征相关;(10)ETV6基因与血小板减少伴肿瘤易感相关;(11)FERMT3基因与白细胞整合素粘附缺陷Ⅲ型相关;(12)GATA1基因与X连锁性血小板减少症伴红细胞生成障碍相关;(13)GFI1B基因与灰色血小板样综合征相关;(14)GNAS基因与血小板缺陷伴智力缺陷相关;(15)GNE基因与血小板减少伴肌病相关;(16)GP1BA基因与巨大血小板综合征(BSS)和血小板型VWD相关相关;(17)GP1BB基因与巨大血小板综合征(BSS)相关;(18)GP6基因与GPVI缺陷相关;(19)GP9基因与巨大血小板综合征(BSS)相关;(20)HOXA11基因与无巨核细胞性血小板减少伴桡尺骨骨性融合相关;(21)HPS1基因与Hermansky-Pudlak综合征相关;(22)HPS3基因与Hermansky-Pudlak综合征相关;(23)HPS4基因与Hermansky-Pudlak综合征相关;(24)HPS5基因与Hermansky-Pudlak综合征相关;(25)HPS6基因与Hermansky-Pudlak综合征相关;(26)ITGA2B与血小板无力症相关;(27)ITGB3与血小板无力症相关;(28)LYST与先天性异常白细胞包涵体CHS综合征相关;(29)MASTL与遗传性血小板减少症2型相关;(30)MPL基因与先天性无巨核细胞血小板减少症(CAMT)相关;(31)MYH9基因与MYH9相关疾病相关;(32)NBEA基因与血小板致密颗粒异常相关;(33)NBEAL2基因与灰色血小板综合征相关;(34)ORAI1基因与Stormorken综合症相关;(35)P2RY12基因与ADP受体缺陷症相关;(36)PLA2G4A基因与IVA型磷脂酶A2缺陷相关;(37)PRKACG基因与血小板型出血性疾病19相关;(38)RASGRP2基因与血小板型出血性疾病18相关;(39)RBM8A基因与血小板减少伴桡骨缺失(TAR)综合症相关;(40)RUNX1基因与家族性血小板紊乱伴AML易感相关;(41)STIM1基因与Stormorken综合症相关;(42)STXBP2基因与家族性噬血细胞性淋巴组织细胞增多症5型相关;(43)TBXA2R基因与血栓素A2受体缺陷相关;(44)TBXAS1基因与Ghosal综合征相关;(45)THPO基因与血小板增多1型相关;(46)TUBB1基因与遗传性巨血小板减少症相关;(47)VIPAS39基因与关节弯曲-肾小管功能不全-新生儿胆汁淤积(ARC)综合症相关;(48)VPS33B基因与关节弯曲-肾小管功能不全-新生儿胆汁淤积(ARC)综合症相关;(49)WAS基因与Wiskott-Aldrich综合征相关。
具体突变位点如表2所示。
2、根据上述基因及突变位点设计引物组合物及其每条引物的浓度为100nM。
3、复合体系PCR扩增:将上述引物混合至一个反应体系中,并选择在保证模板浓度一定的条件下最合适的引物浓度,使复合体系中的每对引物尽量做到最优扩增。
(1)复合扩增体系中的扩增引物
复合扩增体系中,各基因及突变位点的正反向扩增引物如表1所示,为使每个基因及突变位点的扩增效率尽可能的一致,通过调整复合体系中每对引物的浓度,最终调整后的引物浓度为100nM。
(2)DNA提取:利用天根DNA提取试剂盒(货号:DP318-03)提取骨髓样本的全基因组DNA。
(3)根据实施例1提供的引物组合物对提取的DNA进行复合PCR扩增。
其中,复合扩增体系如表3所示。
表3复合扩增体系
成分 | 体积 |
AgriSeqTM Amplification Mix | 2μL |
Ion AmpliSeqTM Primer Pool | 5μL |
稀释后DNA样本(10ng/μL) | 3μL |
DNA来源于所测样本,Ion AmpliSeqTM Primer pool为上述引物的混合物,各引物浓度为100nM,AgriSeqTM Amplification Mix为AgriSeqTM扩增混合物(ThermoFisherScientific,A34144)。
复合扩增反应程序如下:99℃,2min;99℃,15s,62℃,4min,2cycles;99℃,15s,60℃,8min,14cycles;10℃,贮存。
4、采用试剂盒将扩增产物制备成可供测序平台测序的DNA文库,完成建库。
4.1对扩增产物进行酶切。酶切体系如表4所示。
表4酶切体系
步骤3复合扩增产物 | 20μL |
Pre-ligation | 2μL |
酶切反应程序如下:50℃,20min;55℃,20min;60℃,20min;10℃,贮存。反应结束后对产物进行磁珠法纯化。
其中,Pre-ligation购自ThermoFisherScientific,货号为A34144。
4.2末端修复&加A。反应体系如下表5所示。
表5末端修复&加A
酶切产物 | 40μL |
End Repair&A-Tailing Enzyme | 4μL |
End Repair&A-Tailing Buffer | 6μL |
反应程序如下:20℃,30min;65℃,30min;4℃,贮存。
其中,End Repair&A-Tailing Enzyme及End Repair&A-Tailing Buffer购自ThermoFisherScientific,货号A34144。
4.3接头连接。反应体系如下表6所示。
表6接头连接
末端修复&加A产物 | 50μL |
IDT UDI接头(15μM) | 2μL |
Ligation Buffer | 26μL |
DNA Ligase | 2μL |
反应程序如下:20℃,20min。反应结束后对产物进行磁珠法纯化。
其中,IDT UDI接头(15μM)、Ligation Buffer、DNA Ligase购自纳昂达,接头货号1003227,Ligation Buffer和DNA Ligase货号1002103。
4.4文库富集。反应体系如下表7所示。
表7文库富集
2X HiFi PCR Master Mix | 25μL |
扩增引物混合物(10μM) | 5μL |
接头连接纯化后的产物 | 20μL |
扩增反应程序如下:98℃,2min;98℃,15s,60℃,30s,72℃,30s,7cycles;72℃,2min;4℃,贮存。反应结束后对产物进行磁珠法纯化。
其中,2X HiFi PCR Master Mix购自纳昂达,货号1002103。
5.将DNA文库进行测序,得到下机数据,并对下机数据进行生物学分析。
实施例2重复性试验
选取一例经临床症状和相关实验室检查诊断为先天性血小板缺陷的患者,其中,经一代测序检测为WAS基因c.223G>A核苷酸突变,其氨基酸突变为p.V75M。
采用本申请所述的引物组合物及试剂盒重复检测该样本三次,具体步骤及检测方法如下:
1.DNA提取:利用天根DNA提取试剂盒(货号:DP318-03)提取骨髓样本的全基因组DNA。
2.根据实施例1提供的引物组合物及扩增试剂和扩增程序对提取的DNA进行复合多重PCR扩增。
3.将扩增产物制备成可供Ion Torrent测序平台测序的DNA文库,具体操作步骤详见Life公司试剂盒(名称:Ion AmpliSeqTM Library Kit,货号:4480441)的使用说明书。
4.对所获得的DNA文库进行测序,得到下机数据:将构建好的测序文库经油包水处理后在Ion Torrent平台上进行高通量测序,油包水处理方法和高通量测序方法参考高通量测序仪Ion Torrent以及其配套设备One Touch的使用说明书进行。
5.对下机数据进行生物信息学分析:首先,使用Ion Report软件(v4.6,ThermoFisher,Carlsbad,CA,USA)过滤低质量的测序片段,并将合格的测序片段比对到人类参考基因组hg19(http://hgdownload.cse.ucsc.edu/downloads.html),使用Torrent VariantCaller(v4.6.0.7)子程序检测突变位点,软件参数使用的是默认的设置。此软件已经被优化用于处理Ion Torrent测序平台特有的错误类型。最后对找出的突变包括SNP和Indel使用ANNOVAR(http://annovar.openbioinformatics.org/en/latest/)软件进行注释,注释内容包括突变在基因组中的位置,关联的基因,基因外显子编号,核苷酸水平变异,对应的蛋白水平变异,以及该突变在dbSNP数据库(http://www.ncbi.nlm.nih.gov/snp/),千人基因组数据库1000Genomes(http://www.1000genomes.org/),外显子组整合数据库ExAC(http://exac.broadinstitute.org/)和人类基因突变数据库HGMD(http://www.hgmd.cf.ac.uk/ac/index.php)中的注释,PolyPhen(http://genetics.bwh.harvard.edu/pph2/)和SIFT(http://sift.jcvi.org/)功能预测结果等。
得到的结果如下表8所示,三次检测结果均为WAS基因c.223G>A核苷酸突变阳性,表明本申请所述的引物组合物及试剂盒具有较好的重复性。
表8重复性检测结果
检测结果 | 第一次 | 第二次 | 第三次 |
突变基因 | WAS | WAS | WAS |
核苷酸改变 | c.223G>A | c.223G>A | c.223G>A |
氨基酸改变 | p.V75M | p.V75M | p.V75M |
Bed | chrX:48542762 | chrX:48542762 | chrX:48542762 |
测序reads数 | 1379016 | 1377581 | 1379423 |
靶标区域覆盖度 | 99.41% | 98.12% | 99.53% |
平均测序深度(×) | 488 | 472 | 516 |
均一性 | 93.91% | 93.17% | 94.28% |
实施例3特异性试验
共设计三组实验,分别如下:
实验组1、利用实施例1给出的引物及浓度、扩增体系和扩增程序对实施例1提取的DNA样本进行扩增,使用Ion Torrent进行测序,并进一步分析。
实验组2、将实施例1给出的引物浓度均修改为50nM,采用相同的扩增体系和扩增程序对实施例1提取的DNA样本进行扩增,使用Ion Torrent进行测序,并进一步分析。
实验组3、将实施例1给出的引物浓度均修改为200nM,采用相同的扩增体系和扩增程序对实施例1提取的DNA样本进行扩增,使用Ion Torrent进行测序,并进一步分析。
上述实验组1、2、3的检测结果如表9所示。
表9特异性检测结果
检测结果 | 实验组1 | 实验组2 | 实验组3 |
突变基因 | WAS | WAS | WAS |
核苷酸改变 | c.223G>A | c.223G>A | c.223G>A |
氨基酸改变 | p.V75M | p.V75M | p.V75M |
突变位置 | chrX:48542762 | chrX:48542762 | chrX:48542762 |
测序reads数 | 1379016 | 1065194 | 1039826 |
靶标区域覆盖度 | 99.41% | 97.12% | 96.53% |
平均测序深度(×) | 488 | 314 | 309 |
均一性 | 93.91% | 87.64% | 85.41% |
由表9的检测结果可知,引物浓度过低或过高其测序reads数、测序深度及均一性等结果都较差。
实施例4灵敏度试验
根据实施例1给出的引物及浓度和扩增试剂与扩增程序,分别采用实施例2提取的不同浓度的DNA样本进行扩增,使用Ion Torrent进行测序,并进一步分析。其中,DNA模板浓度分别为:5ng/μL、2.5ng/μL、1.25ng/μL、0.625ng/μL、0.3125ng/μL、0.15625ng/μL。
检测结果如表10所示。
表10灵敏度检测结果
由该结果可知:本发明提供的复合扩增体系可以对浓度为0.15625ng/μL的样本进行准确检测,灵敏度为0.64%,远优于一代测序的10%,识别能力强。
实施例5准确性检测
选取在天津见康华美医学诊断中心2016年10月至2021年11月期间经临床症状和相关实验室检查发现血小板减少或功能异常的病例,筛选需要进行基因检测以进行辅助确认是否为先天性相关缺陷患者87例。收集3mL患者外周血提取基因组DNA待测,该检测经过中心伦理委员会批准,并经患者同意。
根据实施例1和实施例2提供试剂及方法进行样本检测,检测结果如下表11所示。
表11准确性检测结果
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由上表可知,上述87例患者中检测到均为先天性血小板缺陷,其中有携带ACBD5基因突变患者1例,携带ACTN1基因突变患者1例,携带ANKRD26基因突变患者8例,携带AP3B1基因突变患者2例,携带BLOC1S6基因突变患者1例,携带ETV6基因突变患者4例,携带GATA1基因突变患者1例,携带GNAS基因突变患者1例,携带GNE基因突变患者2例,携带GP1BA基因突变患者7例,携带GP1BB基因突变患者1例,携带GP6基因突变患者6例,携带HOXA11基因突变患者1例,携带HPS1基因突变患者4例,携带HPS3基因突变患者1例,携带HPS5基因突变患者2例,携带ITGA2B基因突变患者37例,携带ITGB3基因突变患者11例,携带LYST基因突变患者1例,携带MASTL基因突变患者1例,携带MPL基因突变患者4例,携带MYH9基因突变患者22例,携带NBEAL2基因突变患者3例,携带ORAI1基因突变患者1例,携带P2RY12基因突变患者2例,携带PRKACG基因突变患者1例,携带RASGRP2基因突变患者2例,携带RUNX1基因突变患者1例,携带STIM1基因突变患者1例,携带STXBP2基因突变患者2例,携带TBXA2R基因突变患者1例,携带TBXAS1基因突变患者2例,携带TUBB1基因突变患者2例,携带VIPAS39基因突变患者1例,携带WAS基因突变患者11例,而且得出的结论与临床最终诊断相一致。由此可见,使用该检测试剂盒能够有效地对先天性血小板缺陷患者进行诊断,具有诊断快速、准确、获得信息量大的特点。
最后应当说明的是,以上内容仅用以说明本发明的技术方案,而非对本发明保护范围的限制,本领域的普通技术人员对本发明的技术方案进行的简单修改或者等同替换,均不脱离本发明技术方案的实质和范围。
Claims (9)
1.一组检测先天性血小板缺陷基因的突变位点的引物组合物,其特征在于:所述的突变位点位于ACBD5、ACTN1、ANKRD26、ANO6、AP3B1、BLOC1S3、BLOC1S6、CYCS、DTNBP1、ETV6、FERMT3、GATA1、GFI1B、GNAS、GNE、GP1BA、GP1BB、GP6、GP9、HOXA11、HPS1、HPS3、HPS4、HPS5、HPS6、ITGA2B、ITGB3、LYST、MASTL、MPL、MYH9、NBEA、NBEAL2、ORAI1、P2RY12、PLA2G4A、PRKACG、RASGRP2、RBM8A、RUNX1、STIM1、STXBP2、TBXA2R、TBXAS1、THPO、TUBB1、VIPAS39、VPS33B、WAS共49个基因上;所述的引物组合物如下所示
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2.根据权利要求1所述的引物组合物,其特征在于:所述的引物组合物能够扩增权利要求1所述的突变位点中的至少一个。
3.权利要求1或2所述的引物组合物在制备检测先天性血小板缺陷基因的试剂盒中的应用。
4.一种检测先天性血小板缺陷基因的试剂盒,其特征在于:所述的试剂盒包括权利要求1或2所述的引物组合物。
5.根据权利要求4所述的试剂盒,其特征在于:所述的试剂盒还包括将基因组DNA制成可供测序的文库的试剂。
6.根据权利要求4所述的试剂盒,其特征在于:所述的试剂盒还包括用于二代测序的试剂。
7.根据权利要求4所述的试剂盒,其特征在于:所述的试剂盒还包括DNA聚合酶、缓冲液、ddH2O、dNTP。
8.一种检测先天性血小板缺陷基因的方法,其特征在于:所述的方法包括利用权利要求4所述的试剂盒对待测样品中先天性血小板缺陷基因进行检测。
9.根据权利要求8所述的方法,其特征在于:包括以下步骤:
S1、样品基因组DNA提取;
S2、对步骤S1中的DNA进行PCR扩增;
S3、文库构建;
S4、文库测序及数据处理分析。
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