CN114107005A - 一种含有游离脱氧核糖核酸稳定剂的采血管 - Google Patents
一种含有游离脱氧核糖核酸稳定剂的采血管 Download PDFInfo
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Abstract
本发明公开了一种含有游离脱氧核糖核酸稳定剂的采血管,该采血管包括稳定剂、管体、密封塞,所述稳定剂包含防腐剂、抗凝剂、代谢抑制剂、酶抑制剂以及缓冲物质,所述采血管管体由石英、PET塑料等材质制造而成,所述密封胶塞由聚丙烯材料制造而成。本发明采血管中的稳定剂能够有效的防止细胞中基因组DNA释放到血浆中,可以实现常温条件下对血液中游离DNA的稳定保存,有效延长血液样本在室温下的存储时间,且对后续检测结果无影响,可用于实验室、医院等多种医疗生物监测机构,具有良好的使用价值和应用前景。
Description
技术领域
本发明涉及医疗技术领域,特别涉及一种含有游离脱氧核糖核酸稳定剂的采血管。
背景技术
游离DNA(cell free DNA,简称cf-DNA),是指血液中游离于细胞外的DNA,随着cf-DNA分离纯化和鉴定技术日臻完善以及二代基因测序技术的快速发展,对于血液中游离DNA的早期筛查已经成为一种重要的检测手段,它既可以用于基于基因检测技术的癌症的早期筛查、诊断、监控等领域,也可用于无创伤性产前诊断、器官移植和亲子鉴定等基因测序相关产业。然而血液中游离DNA的含量较低,离开人体后易受环境的影响,血样的采集以及保存方式不当时,都会影响cf-DNA的检测分析结果。
目前cf-DNA检测过程中的测序设备昂贵且样本量通常较少,易于降解,存在送检过程中cf-DNA就已经发生降解的状况,另外cf-DNA对于采血管的要求非常高,除了要具备保护cf-DNA不被降解,还需要起到长时间稳定保存的功能。而市面上普通的抗凝采血管对于储存和运输条件要求苛刻,保存时间有限,仅能保存一天左右,不利于需要长时间运输的样本的检测,这些都会造成后续的检测工作无法开展,不能实现对疾病的早发现早治疗。因此,提供一种维持血液中cf-DNA含量长时间稳定保存的采血管是十分必要的。
发明内容
针对现有技术存在的问题,本发明提供了一种含有游离脱氧核糖核酸稳定剂的采血管。本发明采血管中包含的稳定剂能维持血液细胞的稳定性,使血液中cf-DNA能在常温情况下稳定保存28天及以上。
一种含有游离脱氧核糖核酸稳定剂的采血管,包括管体和密封塞,所述采血管管体内还包括DNA稳定剂,所述稳定剂包括缓冲物质、防腐剂、抗凝剂、代谢抑制剂以及酶抑制剂。
进一步,所述缓冲物质为TE Buffer、环糊精、氯化钠、甘氨酸、Tris-HCl中的一种或多种;所述防腐剂为山梨醇、咪唑烷基脲、双(羟甲基)咪唑烷基脲、重氮咪唑烷基脲中的一种或多种;所述抗凝剂为乙二胺四乙酸三钾(EDTA3K)、肝素钠、氟化钠中的一种或多种;所述代谢抑制剂为N-乙基顺丁烯二酰亚胺、溴硝醇、羟基偶氮酯、右旋葡萄糖、鱼藤酮、抗霉素中的一种或多种;所述的酶抑制剂为寡霉素、β-巯基乙醇、金精三羧酸ATA、二硫苏糖醇、2-脱氧葡萄糖(2-DG)中的一种或多种。
进一步,每100ml稳定剂中各组分的含量分别为:所述防腐剂1~5g,所述抗凝剂1~10g,所述代谢抑制剂1~10g,所述酶抑制剂1~5g,每支采血管中所述稳定剂的添加量为20~500ul。
进一步,所述采血管的真空负压容血量为5~10ml。
进一步,所述采血管管体由石英、玻璃、PET塑料等材质制造而成,所述密封塞为聚丙烯制造而成。
进一步,所述采血管用于采集,保存和运输血液,血液采集到所述采血管中,需上下颠倒摇晃10次及以上使其与所述稳定剂充分混合均匀。
进一步,所述稳定剂分装至所述采血管管体中,需经抽真空、辐照灭菌处理。
与现有技术相比,本发明的有益效果是:本发明所述血液游离DNA稳定剂能防止或者减缓游离DNA降解;能够在常温条件下使血液中游离DNA稳定储存长达一个月左右;能够适应长时间的运输条件,为边远地区样本的检测提供了便利。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
附图1为采血管构造示意图。
附图2为血液上下颠倒混匀方法示意图。
附图3为实施例1采血管qPCR检测结果图。
附图4为实施例2采血管qPCR检测结果图。
附图5为实施例3采血管qPCR检测结果图。
附图6为实施例4采血管qPCR检测结果图。
具体实施方式
为了使本发明实施例中的技术方案进行更加清晰,以下结合实施例,对本发明进行更详细的说明。需要指出的是,这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1
稳定剂的制备:
使用校准后的电子天平,称取缓冲物质Tris-HCl 6g,环糊精 3g,甘氨酸 3g;防腐剂咪唑烷基脲 3g;抗凝剂乙二胺四乙酸三钾(EDTA3K)7g;代谢抑制剂N-乙基顺丁烯二酰亚胺3g,右旋葡萄糖3g;酶抑制剂β-巯基乙醇 2g,分别称重后放置于100ml干净的烧杯中,向烧杯中加入100ml去离子水,混合搅拌均匀,放置备用。
采血管的制备:
将配置好的稳定剂按照每支采血管300µl的剂量分装至10ml的石英采血管中,通过抽真空压盖机进行液体负压10ml规格的抽真空处理后,采用15gky辐照灭菌后备用。所述石英采血管可从商业途径获得。
采血管的应用:
使用上述采血管随机抽取血样,每个血样抽取5管,常温25℃,放置0天、7天、14天、21天、28天后进行核酸提取(提取试剂盒为:Qiagen公司QIAamp DNA Kit(50)货号为55128),所采用的引物为健康人体的β-actin基因引物,引物序列为:Forward 5’-AACTACCTTCAACTCCATCA-3’
Reverse 5’-GAGCAATGATCTTGATCTTCA-3’
将所提取的核酸样本进行实时荧光PCR检测,PCR测试结果见图3。与普通EDTA采血管的保存效果进行对比,结果见表1。
表1
测试结果表明,实施例1涉及的稳定剂能够在7天内有效保护和稳定血液中的cf-DNA,cf-DNA仅存在轻微降解,第21天时会发生cf-DNA降解。而常规EDTA采血管对cf-DNA的保护及稳定效果较差,在第7天时降解情况较为严重,后期定量阴性对照组会发生溶血现象,细胞破裂释放基因组DNA,造成无法准确定量cf-DNA。
实施例2
鉴于实施例1中,实施例1配方的稳定剂只能保证21天内血浆中cf-DNA的稳定性,在第28天时,cf-DNA发生了降解,为了解决该问题,我们在实施例2中加入更多种类的核酸酶抑制剂,以抑制cf-DNA被核酸酶降解,稳定cf-DNA的含量。
本实施例2稳定剂的制备如下:
使用校准后的电子天平,称取缓冲物质Tris-HCl 6g,甘氨酸 3g,环糊精 3g;防腐剂咪唑烷基脲 3g;抗凝剂乙二胺四乙酸三钾(EDTA3K)7g;代谢抑制剂N-乙基顺丁烯二酰亚胺3g,右旋葡萄糖3g;酶抑制剂β-巯基乙醇 2g,金精三羧酸ATA 2g,二硫苏糖醇 2g;放置于干净的烧杯中,再向烧杯中加入100ml去离子水,混合搅拌均匀,放置备用。
按照实施例1的步骤制备采血管,并进行样本提取以及实时荧光PCR检测,PCR测试结果见图4。与普通EDTA采血管的保存效果进行对比,结果见表2。
表2
测试结果表明,实施例2涉及的稳定剂的保存效果较实施例1有明显的改善。
实施例3
利用实施例2稳定剂配方制备采血管,随机抽取血样,每个血样抽取5管,使用恒温摇床模拟车载运输条件(25℃,80r/min)。分别在运输0天、7天、14天、21天、28天后进行核酸提取(步骤和试剂同实施例2)。
将所提取的核酸样本进行实时荧光PCR检测,PCR测试结果见图5。同样的用普通的EDTA采血管作为对照,检测结果见表3。
表3
由以上结果可以看出,在模拟车载运输的条件下,本发明中实施例2含稳定剂的采血管,同样可以在28天内保证cf-DNA的稳定性。
实施例4
使用校准后的电子天平,称取缓冲物质Tris-HCl 6g,甘氨酸 3g,环糊精 1g,防腐剂咪唑烷基脲 3g;抗凝剂乙二胺四乙酸三钾(EDTA3K)7g;代谢抑制剂N-乙基顺丁烯二酰亚胺3g,右旋葡萄糖3g,鱼藤酮0.1g;酶抑制剂β-巯基乙醇 2g,寡霉素 1g,二硫苏糖醇 1g,2-DG 1g。
按照实施例1的步骤制备采血管,并进行样本提取以及实时荧光PCR检测,PCR测试结果见图6。与普通EDTA采血管的保存效果进行对比,结果见表4。
表4
由以上结果可以看出,优化代谢抑制剂及核酸酶抑制剂添加种类及剂量的实施例4,在28天内保证cf-DNA的稳定性比实施例2的效果更佳。
所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (7)
1.一种含有游离脱氧核糖核酸稳定剂的采血管,包括管体和密封塞,其特征在于,所述采血管管体内还包括DNA稳定剂,所述稳定剂包括防腐剂、抗凝剂、代谢抑制剂、酶抑制剂以及缓冲物质。
2.根据权利要求1所述的含有游离脱氧核糖核酸稳定剂的采血管,其特征在于,所述缓冲物质为TE Buffer、环糊精、氯化钠、甘氨酸、Tris-HCl中的一种或多种;所述防腐剂为山梨醇、咪唑烷基脲、双(羟甲基)咪唑烷基脲、重氮咪唑烷基脲中的一种或多种;所述抗凝剂为乙二胺四乙酸三钾(EDTA3K)、肝素钠、氟化钠中的一种或多种;所述代谢抑制剂为N-乙基顺丁烯二酰亚胺、溴硝醇、羟基偶氮酯、右旋葡萄糖、鱼藤酮、抗霉素中的一种或多种;所述的酶抑制剂为寡霉素、β-巯基乙醇、金精三羧酸ATA、二硫苏糖醇、2-脱氧葡萄糖(2-DG)中的一种或多种。
3.根据权利要求1所述的含有游离脱氧核糖核酸稳定剂的采血管,其特征在于,每100ml稳定剂中各组分的含量分别为:所述防腐剂1~5g,所述抗凝剂1~10g,所述代谢抑制剂1~10g,所述酶抑制剂1~5g,每支采血管中所述稳定剂的添加量为20~500ul。
4.根据权利要求1所述的含有游离脱氧核糖核酸稳定剂的采血管,其特征在于,所述采血管的真空负压容血量为5~10ml。
5.根据权利要求1所述的含有游离脱氧核糖核酸稳定剂的采血管,其特征在于,所述采血管管体由石英、玻璃、PET塑料等材质制造而成,所述密封塞为聚丙烯制造而成。
6.根据权利要求1-5所述的含有游离脱氧核糖核酸稳定剂的采血管,其特征在于,所述采血管用于采集,保存和运输血液,血液采集到所述采血管中,需上下颠倒摇晃10次及以上使其与所述稳定剂充分混合均匀。
7.根据权利要求1所述的含有游离脱氧核糖核酸稳定剂的采血管,其特征在于,所述稳定剂分装至所述采血管管体中,需经抽真空、辐照灭菌处理。
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US20190254273A1 (en) * | 2016-06-08 | 2019-08-22 | Cfgenome, Llc | A chemical composition to stabilize extracellular vesicles in a blood sample and method of use thereof |
CN107760673A (zh) * | 2017-08-25 | 2018-03-06 | 南通普惠精准医疗科技有限公司 | 游离dna的稳定剂和用于游离dna检测的采血管 |
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