CN112662662A - 一种游离dna保存试剂及制备方法 - Google Patents
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Abstract
本发明公开了一种游离DNA保存试剂,包括乙二胺四乙酸钾盐(EDTA盐)、甲醛、甘氨酸、Tris‑HCL、氯化钠和去离子水;各组分的质量百分比如下:乙二胺四乙酸钾盐1.5‑5%、甲醛0.1‑1%、甘氨酸1‑3%、Tris‑HCL5‑10%、氯化钠1‑20%、去离子水余量。另外,本发明还公开了上述游离DNA保存试剂的制备方法。本发明的游离DNA保存试剂可以保存DNA完整片段,并可实现室温保存至少3‑5天,大大降低了保存和运输成本。
Description
技术领域
本发明属于生命科学和生物技术领域,特别一种游离DNA保存试剂及制备方法。
背景技术
循环游离DNA(cfDNA)是1948年发现的细胞外碎片DNA,主要来源于细胞坏死、凋亡。由肿瘤细胞释放的cfDNA通常称为循环肿瘤DNA(ctDNA),其中一些能反应肿瘤特异性特征,如体细胞突变等,通过检测ctDNA可以很好的代表患者肿瘤的特征,动态监测ctDNA变化,对患者的疾病复发及疗效进行评估等。随着母血中胎儿游离DNA(cell-free fetalDNA,cffDNA)的发现,发展了无创产前筛查(non-invasive prenatal)。这些游离DNA片段大小集中在100-300bp,根据多篇文献报道,一般EDTA抗凝管,采血后都需要尽早分离出血浆,2小时内最佳,6小时已经降解,超过24小时反而含量增加,是因为采血管里的细胞凋亡后重新释放的游离DNA增加,而会稀释原本的游离DNA,尤其是对于无创产筛而言,新凋亡的细胞释放的游离DNA为母亲的游离DNA,会将原本不多的胎儿游离DNA稀释,导致检测失败或假阴性存在。正如前面所述,EDTA抗凝管采集全血后,立即分离出血浆是最佳的,但有的全血样本是要外送检测,立即分离的血浆需要干冰保存运输,这样运输成本大大增加。
发明内容
本发明的第一个目的在于提供一种游离DNA保存试剂,可以保存DNA完整片段,并可实现室温保存至少3-5天,大大降低了保存和运输成本。
一种游离DNA保存试剂,其特征在于,所述游离DNA保存试剂包括乙二胺四乙酸钾盐(EDTA盐)、甲醛、甘氨酸、Tris-HCL、氯化钠和去离子水;各组分的质量百分比如下:
乙二胺四乙酸钾盐 1.5-5%
甲醛 0.1-1%
甘氨酸 1-3%
Tris-HCL 5-10%
氯化钠 1-20%
去离子水余量。
优选地,各组分的质量百分比如下:
更为优选地,各组分的质量百分比如下:
乙二胺四乙酸盐(EDTA盐)能与血液中的Ca离子结合成螯合物,凝血过程被阻断,不能凝血,血液也不能发生凝固。本保存试剂所用的EDTA盐为钾盐,可以为EDTA-K2,也可以为EDTA-K3。在血液中,最佳浓度为1.5mg/ml。EDTA钾盐,溶解度最高,抗凝速度最快。浓度15%的水溶液,每ml血液加1.2mgEDTA,即每5ml血液加0.04ml15%EDTA溶液,不影响白细胞计数,对红细胞形态影响最小,并且可以抑制血小板聚集。若浓度过高则会造成细胞皱缩,另EDTA溶液PH值与盐类关系较大,低PH可使细胞膨胀,可使红细胞体积轻度膨胀,采血后短时间内平均血小板体积非常不稳定,半小时后稳定。最佳浓度为1.5mg/ml血液。甲醛的作用是防腐和固定红细胞,氯化钠可以调节渗透压。
本发明的第二个目的在于提供上述游离DNA保存试剂的制备方法,其特征在于包括以下步骤:将定量的乙二胺四乙酸二钠、Tris、氯化钠、甘氨酸置于烧杯中,加入去离子水溶解,再加入甲醛,用1%盐酸调节pH至5.8~6.8,最后添加去离子水定容至100mL,混合均匀;0.25μm滤膜过滤除菌,即得游离DNA保存试剂。
有益效果:本发明设计了NG淋病奈瑟菌的引物,采用PCR技术,构建了稳定的扩增体系。通过调整引物浓度、退火温度等反应条件,可使扩增效率达到最佳。
附图说明
图1是使用本发明的保存液保存的孕妇外周血5天,文库片段分析图。
具体实施方式
实施例1
按以下质量百分比配制游离DNA保存试剂:
配制方法:将乙二胺四乙酸二钠、Tris、氯化钠、甘氨酸置于烧杯中,加入80%去离子水溶解,再加入甲醛,用1%盐酸调节pH至5.8~6.8,最后添加剩余去离子水定容至100mL,混合均匀;0.25μm滤膜过滤除菌,即得游离DNA保存液。
实施例2
使用实施例1配制的游离DNA保存液,采集孕妇自愿者外周血,常温保存全血游离DNA 5天。使用上述全血,用安诺优达核酸提取试剂和胎儿染色体非整倍体(T21、T18、T13)检测试剂盒进行提取cfDNA,并建库,所得结果及文库片段分析如下:
图1是使用本发明的保存液保存的孕妇外周血5天,文库片段分析图,片段大小符合200bp-300bp片段/200bp-400bp片段比例>70%,且主峰位置<280bp。
Claims (4)
1.一种游离DNA保存试剂,其特征在于,所述游离DNA保存试剂包括乙二胺四乙酸钾盐(EDTA盐)、甲醛、甘氨酸、Tris-HCL、氯化钠和去离子水;各组分的质量百分比如下:
乙二胺四乙酸钾盐 1.5-5%
甲醛 0.1-1%
甘氨酸 1-3%
Tris-HCL 5-10%
氯化钠 1-20%
去离子水余量。
4.权利要求1-3任一所述游离DNA保存试剂的制备方法,其特征在于包括以下步骤:将定量的乙二胺四乙酸二钠、Tris、氯化钠、甘氨酸置于烧杯中,加入去离子水溶解,再加入甲醛,用1%盐酸调节pH至5.8~6.8,最后添加去离子水定容至100mL,混合均匀;0.25μm滤膜过滤除菌,即得游离DNA保存试剂。
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WO2023026725A1 (ja) * | 2021-08-25 | 2023-03-02 | 積水メディカル株式会社 | 細胞含有液用保存液及び細胞含有液用保存容器 |
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US20100184069A1 (en) * | 2009-01-21 | 2010-07-22 | Streck, Inc. | Preservation of fetal nucleic acids in maternal plasma |
CN107267500A (zh) * | 2017-07-17 | 2017-10-20 | 北京安必奇生物科技有限公司 | 一种游离dna保存液及其制备方法和应用 |
WO2018040849A1 (zh) * | 2016-08-30 | 2018-03-08 | 成都瑞琦科技实业股份有限公司 | 孕妇外周血中胎儿游离dna的保存剂及其构成的真空采血管 |
CN108823286A (zh) * | 2018-07-18 | 2018-11-16 | 北京迈基诺基因科技股份有限公司 | 一种血液游离dna的保护剂及真空采血管 |
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US20100184069A1 (en) * | 2009-01-21 | 2010-07-22 | Streck, Inc. | Preservation of fetal nucleic acids in maternal plasma |
WO2018040849A1 (zh) * | 2016-08-30 | 2018-03-08 | 成都瑞琦科技实业股份有限公司 | 孕妇外周血中胎儿游离dna的保存剂及其构成的真空采血管 |
CN107267500A (zh) * | 2017-07-17 | 2017-10-20 | 北京安必奇生物科技有限公司 | 一种游离dna保存液及其制备方法和应用 |
CN108823286A (zh) * | 2018-07-18 | 2018-11-16 | 北京迈基诺基因科技股份有限公司 | 一种血液游离dna的保护剂及真空采血管 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2023026725A1 (ja) * | 2021-08-25 | 2023-03-02 | 積水メディカル株式会社 | 細胞含有液用保存液及び細胞含有液用保存容器 |
JP7310055B1 (ja) * | 2021-08-25 | 2023-07-24 | 積水メディカル株式会社 | 細胞含有液用保存液及び細胞含有液用保存容器 |
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