细胞培养液中细菌、真菌多重荧光定量PCR检测方法的建立及验证  

Development and verification of multiplex fluorescent quantitative PCR for detection of bacteria and fungi in cell culture medium

作　　者：孙锈锈 徐逸梦 周宇荀[1] 李凯[1] 肖君华[1] SUN Xiuxiu;XU Yimeng;ZHOU Yuxun;LI Kai;XIAO Junhua(College of Biological Science and Medical Engineering,Donghua University,Shanghai 201620,China)

机构地区：[1]东华大学生物与医学工程学院,上海201620

出　　处：《中国生物制品学杂志》2024年第3期335-342,共8页Chinese Journal of Biologicals

基　　金：国家自然科学基金(31772550);国家重点研发计划(2018YFA0801101)。

摘　　要：目的建立细胞培养液中常见细菌和真菌的多重荧光定量PCR检测方法,并进行验证。方法选取金黄色葡萄球菌NUC基因、生孢梭菌COLA基因和白色念珠菌ITS-2区段基因,分别设计1组引物及探针,并以辣椒UBI3基因作为外标基因,建立金黄色葡萄球菌、生孢梭菌和外标基因的三重反应体系及白色念珠菌和外标基因的两重反应体系。验证方法的引物特异性、线性范围、检测限、专属性、抗干扰性和精密性。采用建立的方法对100份293T细胞培养液样本进行检测,同时与普通PCR法进行比较。结果3对引物于不同退火温度(56、57、58、59℃)下均可扩增出对应3种菌约100 bp的特异性基因条带,大小与预期相符;3种菌标准品质粒在5.80×10^(6)~5.80×10^(2)copies/μL范围内,与Ct均呈良好的线性关系,标准曲线方程分别为:Y=-3.373 X+37.48、Y=-3.557X+36.59、Y=-3.536 X+39.78,R^(2)均>0.99,扩增效率均在90%~110%范围内;3种菌的检测限均为101CFU/mL;3种菌的引物及探针在易发生交叉反应的6种细胞基因组DNA上无特异性扩增;293T细胞、酵母菌、大肠埃希菌、支原体的基因组DNA对检测无影响;重复性和中间精密性验证CV均<15%。100份细胞培养液样本中,检出14份阳性及86份阴性样本,随机挑选的3个阳性和2个阴性样本普通PCR法检测结果与建立的方法一致。结论建立的用于检测细胞培养液中细菌、真菌的多重荧光定量PCR法具有良好的专属性、抗干扰性和精密性,且简便易操作、成本低、灵敏度高,可快速检测细胞培养过程中的污染情况。Objective To develop and verify a multiplex fluorescent quantitative PCR method for detection of common bacterial and fungal contaminants in cell culture medium.Methods According to NUC gene of Staphylococcus aureus,COLA gene of Clostridium spore,ITS-2 segment sequence of Candida albicans,a set of primers and probes were designed for each respectively,and using UBI3 gene of capsicum introduced as external standard gene,a triple reaction system of Staphylococcus aureus,Clostridium spore and external standard gene and a double reaction system of Candida albicans and external standard gene were established.The primer specificity,linear range,limit of detection,specificity,anti-interference performance and precision of the method were verified.Finally,100 samples of 293T cell culture medium were detected by using the developed method,which was compared with the common PCR method.Results Three pairs of primers all amplified about 100 bp specific gene bands corresponding to the three strains at different annealing temperatures(56,57,58 and59℃),and the size was consistent with the expected.In the range of 5.80×10^(6)—5.80×10^(2) copies/μL,the standard plasmids of the three strains showed a good linear relationship with the Ct values.The standard curve equations were:Y=-3.373 X+37.48,Y=-3.557X+36.59 and Y=-3.536 X+39.78,each R^(2)>0.99,respectively,and the amplification efficiency was in the range of 90%—110%.All the limits of detection of the three strains were 10^(1) CFU/mL.The primers and probes of the three strains showed no specific amplification on the genomic DNA of six kinds of cells that were prone to cross-reaction.The genomic DNA of 293T cells,Yeast,Escherichia coli and Mycoplasma sp.had no effect on the detection.The CVs of repeatability and intermediate precision verification were both less than 15%.Among 100 cell culture medium samples,14 positive and 86 negative samples were detected,and the results of common PCR method for three positive and two negative samples randomly selected were consistent

关 键 词：细胞培养液 细菌 真菌 多重荧光定量PCR 

分 类 号：Q-03[生物学]