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Method and kit for detecting cell DNA content

A detection kit and detection method technology, applied in the detection of cell DNA content, the QPCR method for detecting cell DNA content, to the detection of MDCK cell DNA content and size distribution, quantitative PCR detection, can solve the problem that cannot meet market demand, quantitative Poor accuracy, low detection sensitivity and other problems, to achieve the effect of enriching detection coverage, improving detection rate, high sensitivity and specificity

Active Publication Date: 2022-06-17
BEIJING WANTAI BIOLOGICAL PHARMACY ENTERPRISE
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AI Technical Summary

Problems solved by technology

Due to the difficulties in detection sequence selection, primer probe design and detection system optimization, the detection method used for quantitative detection of residual DNA in MDCK cells and the fragment size

Method used

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  • Method and kit for detecting cell DNA content
  • Method and kit for detecting cell DNA content
  • Method and kit for detecting cell DNA content

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0044]实施例1、引物组的设计

[0045]根据GenBank 中MDCK细胞基因组数据，比对分析序列，查找高丰度的保守区序列，选出同源性较高和占比较多的三段序列（核苷酸序列依次为序列表中SEQ ID NO:10、12、14所示）作为引物设计的目标扩增片段。为了更好的分析监控生物制品的质量，按照检测宿主细胞DNA残留量不低于10ng / 剂、细胞DNA残留的片段最好低于200bp的最低检测要求，设计的3套引物和探针组合物，可以分别检测80-120bp、200-250bp和400bp以上这三种小、中、大扩增片段的MDCK细胞DNA残留情况。利用MEGA 4.0 和Primer Premier 5.0在线引物设计软件，设计出3套引物和探针组合物，引物和探针均由上海百力格生物技术有限公司合成，具体序列情况如下表1所示：

[0046]表1 3套引物和探针的序列

[0047]

[0048]根据以上筛选出的3套引物和探针，可以分别扩增出小、中、大3类扩增产物，这3类扩增产物的核苷酸序列依次为序列表中SEQ ID NO:11、13、15所示。

Example Embodiment

[0049]实施例2、MDCK细胞DNA的检测平台的建立

[0050]2.1、模板的提取

[0051]使用核酸提取试剂盒和核酸提取仪提取MDCK细胞DNA，利用含异硫氰酸胍的裂解液对MDCK细胞破碎裂解，利用磁珠对核酸进行吸附抽提，经过Wash Buffer A和Buffer B洗涤两次，去除蛋白和其它杂质，最终将磁珠与核酸分离，收集溶解在TE缓冲液中，最终的洗脱体积为50µL，即制得50µL含MDCK细胞DNA的提取物，作为定量PCR的扩增模板。取5 µL提取物进行琼脂糖凝胶电泳检测，条带唯一,图1表明3份MDCK细胞DNA提取物纯度良好。

[0052]2.2、标准品的制备

[0053]核酸的最大吸收波长为260nm，在波长260nm时，1OD值相当于双链DNA浓度为50ug / ml。完成MDCK细胞DNA提取后，3份MDCK细胞DNA混合后，先后取样10次，使用紫外可见分光光度计测定DNA溶液的260nm处的吸光值（数值应在0.2～1.0之间），同时测定280nm处吸光值，其中A260 / A280比值应在1.6～1.8之间，以此来计算MDCK细胞DNA的核酸浓度，浓度结果如下表2所示。

[0054]表2.MDCK细胞DNA提取物的OD值测定结果

[0055]

[0056]根据测量的OD260值计算DNA原料浓度均值为27.73ug / ml,相当于MDCK细胞核酸DNA的浓度均值为27.73ng / µL。表2中10次MDCK细胞DNA提取物的浓度测定结果与OD260 / OD280比值的折线图详见图2所示。

[0057]根据测定的MDCK细胞DNA浓度，依次用纯水稀释至终浓度分别为300pg / µL、30pg / µL、3pg / µL、0.3pg / µL、0.03pg / µL和0.003pg / µL，以此作为6个已知浓度的定量标准品。

[0058]2.3、反应体系

[0059]3套引物和探针组合物分别设置在3个单独的PCR扩增管中独立进行MDCK细胞DNA的定量PCR检测，每套引物和探针组合物所在管均制备40µL定量PCR反应体系，包括DNA、dNTP混合物、Mg2+离子、Taq DNA聚合酶、正向引物(15uM)、反向引物(15uM)、探针(10uM)、10×PCR Buffer和MDCK细胞DNA标准品，其中、2.5mM dNTP混合物4µL、4U Taq DNA聚合酶0.4µL、正向引物和反向引物...

Example Embodiment

[0062]实施例3、MDCK细胞DNA的反应条件的优化

[0063]3.1、Mg2+浓度调试

[0064]按上述40µL的反应体系进行准备，稀释MDCK细胞DNA至3pg / µL作为待测样本检测3孔重复为1组，NC为无样本的阴性对照，每组均包含三套引物探针片段独立配制反应体系，平行设置2组，第1组反应体系中不含Mg2+，第2组额外添加2µL的50mM的Mg2+（终浓度为2.5mM），将上述准备好的2组反应管置于荧光定量PCR仪中进行QPCR反应，Mg2+浓度调试实验的扩增曲线图如图3所示。图3中的A、B、C分别代表了小片段、中片段和大片段的引物和探针组合物对应的扩增曲线图，NC代表的阴性对照为一条直线，1代表第1组不含Mg2+的反应体系的扩增曲线，2代表第2组含2.5mM Mg2+的反应体系的扩增曲线，图3中明显可见第2组含2.5mM Mg2+的反应体系的标准曲线的可以获得相对较小的Ct值，Ct值较小表明达到检测阈值的循环数越少，荧光信号能够很快达到设定阈值，因此，扩增效率更高。

[0065]3.2、退火延伸温度优化

[0066]按上述优化的40µL的反应体系（含2.5mM Mg2+）进行准备，准备待测样本Vero、U2、CHO、E.coil、赤毕酵母和大鼠基因组的DNA样本，待测样本分别配制于3套引物和探针对应的PCR反应体系中，每份待测样本设置2孔重复为1组，每份重复分别在反应程序1和反应程序2中同步进行检测。

[0067]反应程序1：第一步：95℃预变性5分钟；第二步：95℃变性15秒，60℃退火延伸90秒，延伸后采集FAM荧光；重复运行第二步40个循环。

[0068]反应程序2：第一步：95℃预变性5分钟；第二步：95℃变性15秒，56℃退火延伸90秒，延伸后采集FAM荧光；重复运行第二步40个循环。

[0069]以上2组反应程序的区别之处在于退火延伸温度不同，扩增结果如下表3所示，N / A表示无扩增，检测结果为阴性，数值为检测Ct值表示有扩增，检测结果为阳性。

[0070]表3 退火延伸温度优化对比试验

[0071]

[0072]从表3扩增结果可以看出：退火温度为60℃时，3套引物对6种非靶标样本的特异性最好，没有出现假阳性扩增结果，而当退火温度为56℃时，3套引物均出现个别非特异性起跳,即出现假阳性的检测结果。

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Abstract

The invention provides a primer and probe composition, a detection method for simultaneously detecting the content and size distribution of cell DNA fragments and a kit thereof, the primer and probe composition comprises three sets of primers and probes, a 40L quantitative PCR reaction system is optimally arranged in the detection reaction, the annealing extension temperature in the reaction procedure is 60 DEG C, and the temperature is 30 DEG C; the three sets of primers and probes independently detect small, medium and large DNA fragments of the same sample in a single tube, the sensitivity can reach 0.003 pg/L, the false positive reaction is effectively avoided, the specificity is high, the size distribution of MDCK cells can be analyzed at the same time, and the size distribution is far higher than the conventional standard requirement.

Description

technical field [0001] The invention relates to the technical field of nucleic acid detection methods, in particular to a quantitative PCR (QPCR) detection method, in particular to a QPCR method for detecting cell DNA content; the present invention also particularly relates to a method for detecting cell DNA content and its The kit can be used in the detection of cell DNA content and size distribution, especially the detection of MDCK cell DNA content and size distribution. Background technique [0002] Residual host cell DNA in biological products derived from cell culture is a potential safety risk. Therefore, both product production and drug supervision need to confirm the degree of removal of cellular DNA by the process, especially the detection of residual cellular DNA content in the final product is particularly important. The three methods published in the 2020 edition of "Chinese Pharmacopoeia": PicoGreen fluorescence method, DNA probe hybridization method and quanti...

Claims

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Application Information

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IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686C12Q2545/114
Inventor 李珂马万里贾继宗韩金乐叶祥忠
Owner BEIJING WANTAI BIOLOGICAL PHARMACY ENTERPRISE