摘要
研制大肠埃希菌DNA残留量测定所用质控品.抽提大肠埃希菌基因组DNA作为质控品原料,通过紫外分光光度法和电泳进行纯度和含量测定,采用紫外分光光度法和定量PCR法检测其均匀性和稳定性,并评价其加标回收率.经检测,A260/A 280均在1.8～2.0之间,电泳图谱条带单一,无RNA和寡核苷酸存在.应用紫外分光光度法和定量PCR法对质控品进行均匀性和稳定性检测,统计结果表明,质控品均匀性良好;在-20℃的保存条件下,半年内未发现不稳定现象;在25℃的保存条件下,7 d内未发现不稳定现象.进行定量PCR法测定,在10-2～103 pg之间线性良好,标准曲线R值为0.997,斜率为-3.65,同时每组加标样品的回收率在70％～130％之间,SRSD≤20％.该批大肠杆菌DNA质控品各项指标均符合要求,可作为质控品用于定量PCR检测大肠埃希菌的DNA残留量,含量定为111.73 μg/mL.
Abstract
The paper aims to prepare the quality control for quantitative determination of residual DNA in E.coil.Extracting genomic DNA of Escherichia coli as a quality control material and analyzed for purity and concentration by UV spectrophotometry and agarose gel electrophoresis,the homogeneity and stability were checked by UV spectrophotometry and real-time PCR and evaluate its spiked recovery rate.The prepared quality control of Escherichia coli DNA was qualified as indicated by A260/A280 between 1.8 and 2.0 and a single specific band in agarose gel electrophoresis without RNA or oligonucleotides.The result showed the quality control materials were homogenous and stable under the-20 ℃ during 6 month's stock and 25 ℃ during 7 day's stock.The real-time PCR had high sensitivity up to 10-2-103 pg/μL of DNA with good linearity(r=0.997)with a slope of-3.65,and the recovery rate of each group of spiked samples was between 70％and 130％with the SRSD was less than or equal 20％.The prepared quality control was qualified in overall tests with DNA concentration was 111.73 µg/mL and may be used as quality control for residual DNA assay by real-time PCR methods.
基金项目
南京市市场监督管理局科技项目(Kj2020021)
&&(Kj2020023)
&&(Kj2019022)
维普
万方数据