中国药科大学学报2025，Vol.56Issue(2)：176-182,7.DOI:10.11665/j.issn.1000-5048.2024043002
实时荧光定量PCR法检测左旋多巴中大肠埃希菌宿主细胞DNA残留量
Detection of residual DNA in host cells of Escherichia coli in levodopa by Real-time PCR
许冰瑜 1刘妍 2郭心瑶 1严方 1孙桂斌3
作者信息
- 1. 中国药科大学药物分析系,南京 210009
- 2. 哈尔滨工业大学医院药局,哈尔滨 150000
- 3. 南京希罗斯医药有限公司,南京 210038
- 折叠
摘要
Abstract
Using Real-time PCR technology,a highly specific and sensitive method for detecting DNA residues of Escherichia coli host cells in levodopa was established,validated,and preliminarily applied.Escherichia coli strain MB6 16S ribosomal RNA gene was selected as the target gene to design multiple pairs of primers and the target fragment by specific amplification of PCR was obtained.The target fragment was cloned into the pLENTI-BSD-CON vector and the recombinant plasmid was constructed and named pLENTI-BSD-CON-E.coli-16S.A quantitative PCR detection method(SYBR Green method)with magnetic bead extraction and purification methods was established with the reference standard of the recombinant plasmid.Furthermore,the established method was validated,including linear and range,accuracy,precision,specificity,and quantification limit,and applied to the detection of levodopa raw materials.Meanwhile,the detection method was compared with the Taqman probe method by the commercial kit.The primer sequences of the quantitative PCR detection method(SYBR Green method)were TTCGATGCAACGCGAAGAAC(forward)and GTGTAGCCCTGGTCGTAAGG(reverse).The standard curve of DNA was in the range of 10 fg/μL to 3 ng/μL with good linearity(R2≥0.98).The quantitative limit was 10 fg/μL.In addition,the detection recovery rate was in the range of 59.7%to 80.7%,with RSD at less than 15%.Nine batches of levodopa were detected by this method,and the amount of E.coli DNA residue was below the limit.The developed qPCR method can be used for quantitative detection of residual DNA in biological products produced by E.coli as host cells,such as levodopa.The results indicate that the sensitivity of the detection method for recombinant plasmid construction standards is superior than the reagent kit detection method.关键词
大肠埃希菌/左旋多巴/构建质粒/实时荧光定量PCR/外源性DNA残留/帕金森病Key words
E.coli/levodopa/plasmid construction/Real-time PCR/residual exogenous DNA/Parkinson's disease分类
药学引用本文复制引用
许冰瑜,刘妍,郭心瑶,严方,孙桂斌..实时荧光定量PCR法检测左旋多巴中大肠埃希菌宿主细胞DNA残留量[J].中国药科大学学报,2025,56(2):176-182,7.
