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

作　　者：许冰瑜 刘妍[2] 郭心瑶 严方[1] 孙桂斌 XU Bingyu;LIU Yan;GUO Xinyao;YAN Fang;SUN Guibin(Department of Pharmaceutical Analysis,China Pharmaceutical University,Nanjing 210009;Department of Pharmacy,Harbin Institute of Technology Hospital,Harbin 150000;Nanjing Xiluosi Pharmaceutical Co.,Ltd.,Nanjing 210038,China)

机构地区：[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

出　　处：《中国药科大学学报》2025年第2期176-182,共7页Journal of China Pharmaceutical University

摘　　要：采用实时荧光定量PCR技术,建立特异性强、灵敏度高的左旋多巴中大肠埃希菌(Escherichiacoli)宿主细胞DNA残留量的检测方法,并进行验证和初步应用。以大肠埃希菌中的相对保守的E.coli MB6菌株16S核糖体RNA基因序列作为目的基因设计多对引物,通过PCR进行特异性扩增获得目的片段。将目的片段重组至pLENTI-BSD-CON载体构建重组质粒命名为pLENTI-BSD-CON-E.coli-16S,以其为标准品,结合磁珠法提取纯化DNA,建立定量PCR检测方法(SYBR-Green法)。对建立的方法进行线性与范围、准确度、精密度、专属性、定量限及耐用性的方法学验证,应用于左旋多巴原料药的检测,并与试剂盒检测方法(Taqman探针法)进行比较。建立的定量PCR检测方法(SYBR-Green法)正向引物序列:5'-TTCGATGCAACGCGAAGAAC-3';反向引物序列:5'-GTGTAGCCCTGGTCGTAAGG-3'。以重组质粒作为标准品,DNA质量浓度在10fg/μL~3 ng/μL范围内线性关系良好(R^(2)≥0.98),定量限为10 fg/μL,加标溶液回收率在59.7%~80.7%范围内,RSD均小于30%。应用该法对3批左旋多巴原料药进行检测,DNA残留量均在限度以下。结果表明,建立的检测方法可用于定量检测左旋多巴等由大肠埃希菌作为宿主细胞生产的生物制品的DNA残留量,并且其灵敏度优于试剂盒检测方法。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 MB616S 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 pLENTIBSD-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(R^(2)≥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 

分 类 号：R917[医药卫生—药物分析学]