CN114107481A - 一种检测胎儿伴性遗传病的试剂和试剂盒 - Google Patents
一种检测胎儿伴性遗传病的试剂和试剂盒 Download PDFInfo
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Abstract
本发明公开了一种检测胎儿伴性遗传病的试剂和试剂盒。本发明检测胎儿伴性遗传病的试剂,包括Y染色体特异性引物和Y染色体特异性探针,Y染色体特异性引物的上游引物为Seq ID No.1,下游引物为Seq ID No.2所示序列,Y染色体特异性探针为Seq ID No.3所示序列的5’端具有荧光基团、3’端具有淬灭基团的探针。本发明检测胎儿伴性遗传病的试剂,通过检测孕妇血液就可以判断胎儿性别,实现伴性遗传病的检测;可以在孕早期尽早发现和胎儿性别相关的伴性遗传病,为相关治疗提供参考。
Description
技术领域
本发明涉及胎儿早期检测领域,特别是涉及一种检测胎儿伴性遗传病的试剂和试剂盒。
背景技术
胎儿伴性遗传病是指与胎儿性别相关的遗传病,例如先天性丙种球蛋白血症、X连锁型脑水肿或智力低下等。其中,X染色体隐性遗传和伴Y染色体遗传,可以直接通过检测胎儿的性别实现判断。
但是,传统的胎儿性别检测方法主要是通过四维或彩超进行,临床上需要胎儿成长到一定程度才能准确判断,这对孕妇极其不利。并且,通过四维或彩超判断胎儿性别的准确率只有85%左右,不利于胎儿伴性遗传病的检测。
此外,还可以通过羊水穿刺等有创检测方法进行胎儿性别检测;但是,一般产检过程中都避免采用有创检测,毕竟有创检测存在安全隐患。特别是对于一些特殊群体,例如40岁以上的高龄产妇、具有多次流产史的产妇等,有创检测的风险将会极大的提高。
更为重要的是,现有的胎儿性别检测方法或者胎儿伴性遗传病诊断技术,都很难在孕早期,即怀孕12周之前,进行准确有效的检测。
发明内容
本发明的目的是提供一种新的检测胎儿伴性遗传病的试剂和试剂盒。
为了实现上述目的,本发明采用了以下技术方案:
本发明公开了一种检测胎儿伴性遗传病的试剂,包括Y染色体特异性引物和Y染色体特异性探针,所述Y染色体特异性引物的上游引物为Seq ID No.1,下游引物为Seq IDNo.2所示序列,所述Y染色体特异性探针为Seq ID No.3所示序列的5’端具有荧光基团、3’端具有淬灭基团的探针;
Seq ID No.1:5’-AGACCAAGGAATGACCA-3’
Seq ID No.2:5’-CAGGATTGCTGAGGTG-3’
Seq ID No.3:5’-CATGATGCCCCACGGC-3’。
本发明的试剂中,所述Y染色体特异性引物是针对性染色体Y设计的PCR扩增引物,所述Y染色体特异性探针是在Y染色体特异性引物的PCR扩增靶标范围内的,特异性结合性染色体Y的探针。采用本发明的Y染色体特异性引物和Y染色体特异性探针进行实时荧光定量PCR扩增,能够准确判断胎儿的性别,从而为胎儿伴性遗传病检测提供科学参考。更重要的是,本发明的试剂最早能够对最早六孕周的孕妇的血液进行检测,实现孕早期的无创胎儿伴性遗传病检测。
优选的,本发明的试剂还包括看家基因特异性引物和看家基因特异性探针,所述看家基因特异性引物的上游引物为Seq ID No.4所示序列,下游引物为Seq ID No.5所示序列,所述看家基因特异性探针为Seq ID No.6所示序列的5’端具有荧光基团,3’端具有淬灭基团的探针;
Seq ID No.4:5’-ACGACCACTTTGTCAA-3’
Seq ID No.5∶5’-GCCAGACCCTGCACTTT-3’
Seq ID No.6:5’-GGCTGGGGCCAGAGACT-3’。
本发明的试剂中,所述看家基因特异性引物和看家基因特异性探针主要是为了对看家基因进行特异性检测,作为对照,判断检测过程的准确性和有效性。
优选的,所述Y染色体特异性探针的荧光基团为FAM,淬灭基团为BHQ2。
优选的,所述看家基因特异性探针的荧光基团为HEX,淬灭基团为BHQ2。
本发明还公开了本发明的试剂在制备胎儿性别鉴定、受精胚胎性别鉴定、胎儿伴性遗传病检测或受精胚胎伴性遗传病检测的试剂盒中的应用。
本发明还公开了一种胎儿伴性遗传病的检测试剂盒,所述试剂盒中含有本发明的试剂。
优选的,所述检测试剂盒中还含有探针法实时荧光定量PCR扩增缓冲液。
优选的,所述检测试剂盒中还含有血浆游离DNA提取试剂。
由于采用以上技术方案,本发明的有益效果在于:
本发明检测胎儿伴性遗传病的试剂和试剂盒,能够对孕早期,至少六孕周的孕妇的血液中的游离DNA进行检测,实现了孕早期胎儿伴性遗传病的无创安全检测,检测准确率高、成本低,可以在孕早期尽早发现和胎儿性别相关的伴性遗传病。
具体实施方式
胎儿性别鉴定是有效预防伴性遗传病的重要手段,越早发现,对孕妇的影响伤害越小。本发明检测胎儿伴性遗传病的试剂,实际上就是针对孕妇游离DNA中存在的胎儿游离DNA进行检测,以此判断胎儿性别,结果不仅更准确,而且没有任何流产风险。
孕妇血液中的胎儿游离DNA是胎儿的细胞代谢释放的DNA,含量较低;特别是孕早期,含量尤其少。但是,本发明的试剂能够准确的对最早孕六周的孕妇进行胎儿性别及胎儿伴性遗传病检测,大大提早了能够准确鉴别胎儿性别的孕周。并且,本发明进行检测所需的血液量较少,例如本发明的实施例中只使用了100μL孕妇血液,即可完成检测。
下面通过具体实施例对本发明作进一步详细说明。以下实施例仅对本发明进行进一步说明,不应理解为对本发明的限制。
实施例
一、引物和探针的设计
针对Y染色体和看家基因分别设计特异性的检测引物和探针,具体序列见表1,所有引物和探针由上海生工合成。
表1引物和探针
编号 | 序列(5’→3’) | Seq ID No. |
Y-F | AGACCAAGGAATGACCA | 1 |
Y-R | CAGGATTGCTGAGGTG | 2 |
Y-P | 5’FAM-CATGATGCCCCACGGC-3’BHQ2 | 3 |
G-F | ACGACCACTTTGTCAA | 4 |
G-R | GCCAGACCCTGCACTTT | 5 |
G-P | 5’HEX-GGCTGGGGCCAGAGACT-3’BHQ2 | 6 |
表1中“Y”开头的是Y染色体特异性引物和Y染色体特异性探针,“G”开头的是看家基因特异性引物和看家基因特异性探针。“F”表示上游引物,“R”表示下游引物,“P”表示探针。Y染色体特异性探针的荧光基团为FAM,淬灭基团为BHQ2,看家基因特异性探针的荧光基团为HEX,淬灭基团为BHQ2。
二、孕妇血浆游离DNA提取
采用6孕周及以上的孕妇的血浆100μL,用Qiagen血浆游离DNA抽提试剂盒抽提孕妇血浆游离DNA,产物采用20μL AE复溶。
本试验分别采用了孕周为6-12的孕妇的血浆进行试验,总计88例。
三、胎儿性别检测
用takara公司的Premix Ex TaqTM(RR390A)试剂盒进行TaqMan实时荧光检测。
反应体系:2×Premix Ex Taq MIX 25μL、10μM的Y-F和Y-R混合液1μL、10pM的G-F和G-R混合液1μL、10pM的Y-P 1μL、10pM的G-P 1μL、血浆游离DNA 5μL,余量为ddH2O,总计50μL。
反应条件:98℃1min,“95℃5s、58℃30s”循环40次。
四、结果及分析
88个样本的检测结果显示,其中50个样本具有FAM通道的检测信号,Ct值在24-32之间,HEX通道的检测信号Ct值在25-35之间;另外38个样本没有FAM通道的检测信号,只有HEX通道的检测信号,且Ct值在25-35之间。因此,88个样本有50个样本为男胎,可能存在Y染色体遗传相关的遗传疾病;另外38个样本为女胎,可能存在X染色体隐性遗传相关的遗传疾病。对比88个样本胎儿出生后的性别,结果显示,本试验的检测试剂能够准确检测胎儿性别,与实际情况的一致性达到100%。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
序列表
<110> 深圳知因细胞生物科技有限公司
<120> 一种检测胎儿伴性遗传病的试剂和试剂盒
<130> 20211112
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Claims (8)
1.一种检测胎儿伴性遗传病的试剂,其特征在于,包括Y染色体特异性引物和Y染色体特异性探针,Y染色体特异性引物的上游引物为Seq ID No.1,下游引物为Seq ID No.2所示序列,Y染色体特异性探针为Seq ID No.3所示序列的5’端具有荧光基团、3’端具有淬灭基团的探针;
Seq ID No.1:5’-AGACCAAGGAATGACCA-3’
Seq ID No.2:5’-CAGGATTGCTGAGGTG-3’
Seq ID No.3:5’-CATGATGCCCCACGGC-3’。
2.根据权利要求1所述的试剂,其特征在于,还包括看家基因特异性引物和看家基因特异性探针,所述看家基因特异性引物的上游引物为Seq ID No.4所示序列,下游引物为SeqID No.5所示序列,所述看家基因特异性探针为Seq ID No.6所示序列的5’端具有荧光基团,3’端具有淬灭基团的探针;
Seq ID No.4:5’-ACGACCACTTTGTCAA-3’
Seq ID No.5:5’-GCCAGACCCTGCACTTT-3’
Seq ID No.6:5’-GGCTGGGGCCAGAGACT-3’。
3.根据权利要求2所述的试剂,其特征在于,所述Y染色体特异性探针的荧光基团为FAM,淬灭基团为BHQ2。
4.根据权利要求3所述的试剂,其特征在于,所述看家基因特异性探针的荧光基团为HEX,淬灭基团为BHQ2。
5.根据权利要求1-4任一项所述的试剂在制备胎儿性别鉴定、受精胚胎性别鉴定、胎儿伴性遗传病检测或受精胚胎伴性遗传病检测的试剂盒中的应用。
6.一种胎儿伴性遗传病的检测试剂盒,其特征在于,所述检测试剂盒中含有权利要求1-4任一项所述的试剂。
7.根据权利要求6所述的检测试剂盒,其特征在于,所述检测试剂盒中还含有探针法实时荧光定量PCR扩增缓冲液。
8.根据权利要求6或7所述的检测试剂盒,其特征在于,所述检测试剂盒中还含有血浆游离DNA提取试剂。
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