CN115248312A - 一种检测血清dna的方法 - Google Patents
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Abstract
本发明公开了一种检测血清DNA的方法，包括以下步骤：S1、包被：用包被缓冲液将单克隆抗体浓度稀释；S2、封闭：加入封闭液，1h后用缓冲液洗涤；S3、抗原抗体反应：封闭完成后，相应的反应管中加入标准品或检测样品；S4、抗体结合：抗原抗体反应完成后，每个反应管加入抗体；S5、链亲和素结合：抗体结合完成后，每个反应管加入链亲和素；S6、DNA结合：链亲和素结合完成后，每个反应管加入DNA；S7、实时荧光定量PCR反应：以洗涤完成后每个反应管所得DNA为模板分别进行实时荧光定量PCR反应；S8、确定检测样品的DNA含量。通过上述方式，本发明能够为肿瘤患者血清学诊断和治疗前后的连续检测、预后，提供实验检测依据。
Description
技术领域
本发明属于血清学检测技术领域，特别是涉及一种检测血清DNA的方法。
背景技术
血清或血浆cfDNA的大小在166bp左右，其释放入血的生物机制尚未被完全揭示，目前认为主要来自细胞凋亡，其他来源包括坏死肿瘤细胞的溶解、白细胞的分解、新合成核酸的自主释放、细菌病毒等病原体的分解等。cfDNA在血液与蛋白质或膜结合颗粒形成天然复合物，能抵抗核酸酶的降解。正常健康人群的cfDNA水平较低，一般在0～100ng/mL，但在某些病理情况下会显著升高，比如恶性肿瘤、手术和创伤、中风、心衰、自身免疫性疾病、胰腺炎、重症监护相关状况、运动员大量训练后等。癌症患者与健康个体相比有较高的cfDNA水平。这些升高的cfDNA主要来自癌细胞。近年来，随着DNA提取、检测、测序等分子生物学技术发展，使得cfDNA的研究取得了长足进步，与疾病之间的关系逐渐被揭示出来。cfDNA作为生物标志物，在疾病筛查、诊断、治疗监控、预后、疾病复发等方面具有广泛应用。
发明内容
本发明主要解决的技术问题是提供一种检测血清DNA的方法，为肿瘤患者血清学诊断和治疗前后的连续检测、预后，提供实验检测依据。
为解决上述技术问题，本发明采用的一个技术方案是：提供一种检测血清DNA的方法，包括以下步骤：
S1、包被：用包被缓冲液将单克隆抗体浓度稀释至10μg/ml，然后加入到实时荧光定量PCR反应管中，每个反应管加入30μl，37℃，1h后用缓冲液洗涤；
S2、封闭：加入封闭液，37℃，1h后用缓冲液洗涤；
S3、抗原抗体反应：封闭完成后，相应的反应管中加入30μl的标准品或检测样品，4℃过夜，弃去液体，然后用缓冲液洗涤；
S4、抗体结合：抗原抗体反应完成后，每个反应管加入30μl的抗体，37℃，1h后用缓冲液洗涤；
S5、链亲和素结合：抗体结合完成后，每个反应管加入30μl的链亲和素，37℃，30min，缓冲液洗涤；
S6、DNA结合：链亲和素结合完成后，每个反应管加入30μl的DNA，37℃，30min后用缓冲液洗涤；
S7、实时荧光定量PCR反应：以洗涤完成后每个反应管所得DNA为模板分别进行实时荧光定量PCR反应；
S8、确定检测样品的DNA含量：根据标准曲线，按照每孔检测样品的循环数确定其DNA含量。
进一步地说，步骤S7中的PCR反应条件为：94℃预变性5min，94℃变性45s，60℃退火30s，72℃延伸30s；35～40个循环结束后于72℃延伸10分钟。
进一步地说，步骤S2中的封闭液为5mg/ml BSA。
进一步地说，步骤S2中的所述的标准品为梯度浓度稀释的抗体标准品；所述的检测样品为待测血清。
本发明的有益效果是：本发明能够为肿瘤患者血清学诊断和治疗前后的连续检测、预后，提供实验检测依据。
具体实施方式
下面对本发明的较佳实施例进行详细阐述，以使本发明的优点和特征能更易于被本领域技术人员理解，从而对本发明的保护范围做出更为清楚明确的界定。
实施例：一种检测血清DNA的方法，本发明包括以下步骤：
S1、包被：用包被缓冲液将单克隆抗体浓度稀释至10μg/ml，然后加入到实时荧光定量PCR反应管中，每个反应管加入30μl，37℃，1h后用缓冲液洗涤；
S2、封闭：加入封闭液，37℃，1h后用缓冲液洗涤；
S3、抗原抗体反应：封闭完成后，相应的反应管中加入30μl的标准品或检测样品，4℃过夜，弃去液体，然后用缓冲液洗涤；
S4、抗体结合：抗原抗体反应完成后，每个反应管加入30μl的抗体，37℃，1h后用缓冲液洗涤；
S5、链亲和素结合：抗体结合完成后，每个反应管加入30μl的链亲和素，37℃，30min，缓冲液洗涤；
S6、DNA结合：链亲和素结合完成后，每个反应管加入30μl的DNA，37℃，30min后用缓冲液洗涤；
S7、实时荧光定量PCR反应：以洗涤完成后每个反应管所得DNA为模板分别进行实时荧光定量PCR反应；
S8、确定检测样品的DNA含量：根据标准曲线，按照每孔检测样品的循环数确定其DNA含量。
步骤S7中的PCR反应条件为：94℃预变性5min，94℃变性45s，60℃退火30s，72℃延伸30s；35～40个循环结束后于72℃延伸10分钟。
步骤S2中的封闭液为5mg/ml BSA。
步骤S2中的所述的标准品为梯度浓度稀释的抗体标准品；所述的检测样品为待测血清。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书所作的等效结构变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。
Claims (4)
1.一种检测血清DNA的方法，其特征在于：包括以下步骤：
S1、包被：用包被缓冲液将单克隆抗体浓度稀释至10μg/ml，然后加入到实时荧光定量PCR反应管中，每个反应管加入30μl，37℃，1h后用缓冲液洗涤；
S2、封闭：加入封闭液，37℃，1h后用缓冲液洗涤；
S3、抗原抗体反应：封闭完成后，相应的反应管中加入30μl的标准品或检测样品，4℃过夜，弃去液体，然后用缓冲液洗涤；
S4、抗体结合：抗原抗体反应完成后，每个反应管加入30μl的抗体，37℃，1h后用缓冲液洗涤；
S5、链亲和素结合：抗体结合完成后，每个反应管加入30μl的链亲和素，37℃，30min，缓冲液洗涤；
S6、DNA结合：链亲和素结合完成后，每个反应管加入30μl的DNA，37℃，30min后用缓冲液洗涤；
S7、实时荧光定量PCR反应：以洗涤完成后每个反应管所得DNA为模板分别进行实时荧光定量PCR反应；
S8、确定检测样品的DNA含量：根据标准曲线，按照每孔检测样品的循环数确定其DNA含量。
2.根据权利要求1所述的一种检测血清DNA的方法，其特征在于：步骤S7中的PCR反应条件为：94℃预变性5min，94℃变性45s，60℃退火30s，72℃延伸30s；35～40个循环结束后于72℃延伸10分钟。
3.根据权利要求1所述的一种检测血清DNA的方法，其特征在于：步骤S2中的封闭液为5mg/mlBSA。
4.根据权利要求1所述的一种检测血清DNA的方法，其特征在于：步骤S2中的所述的标准品为梯度浓度稀释的抗体标准品；所述的检测样品为待测血清。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117031004A (zh) * | 2023-08-09 | 2023-11-10 | 四川省医学科学院·四川省人民医院 | 一种检测网膜素-1的试剂在制备预测或评估ibs患者生活质量的产品中的应用 |
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| CN117031004A (zh) * | 2023-08-09 | 2023-11-10 | 四川省医学科学院·四川省人民医院 | 一种检测网膜素-1的试剂在制备预测或评估ibs患者生活质量的产品中的应用 |
| CN117031004B (zh) * | 2023-08-09 | 2024-10-01 | 四川省医学科学院·四川省人民医院 | 一种检测网膜素-1的试剂在制备预测或评估ibs患者生活质量的产品中的应用 |
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