两种核酸扩增检测方法用于病毒载体制品支原体检测的比较  

Comparative study of two nucleic acid amplification detection methods for mycoplasma detection of viral vector products

作　　者：苏文浩 黄秋芳 任秀秀 赵婷婷 王轶男 李实实 王晓杰 张晓焕 卫江波 Su Wenhao;Huang Qiufang;Ren Xiuxiu;Zhao Tingting;Wang Yinan;Li Shishi;Wang Xiaojie;Zhang Xiaohuan;Wei Jiangbo(Herpesvirus Research Group,National Vaccine and Serum Institute,Beijing 101111,China)

机构地区：[1]国药中生生物技术研究院有限公司疱疹课题组,北京101111

出　　处：《国际生物制品学杂志》2022年第4期210-216,共7页International Journal of Biologicals

基　　金：国家科技重大专项"重大新药创制"(2013ZX09402302-224)。

摘　　要：目的研究两种支原体快速检测方法在病毒载体制品中的适用性和影响因素。方法采用基于PCR技术的两种支原体核酸扩增检测方法,对分别表达Ⅱ型单纯疱疹病毒抗原gD和ICP27的重组仙台病毒载体疫苗(SeV-dF/HSV-2 gD、SeV-dF/HSV-2 ICP27)收获液及纯化液进行支原体检测,探究其特异性和最低检测限,并对多个批次的收获液及纯化液进行测定。结果普通PCR法在对本制品病毒收获液检测时无明显PCR抑制现象,但对病毒纯化液检测时发生明显的PCR抑制,并且未能检出100 CFU/ml限度的口腔支原体和肺炎支原体。实时荧光定量PCR法在对本制品病毒收获液和纯化液的支原体检测中均无PCR抑制发生,且该方法对口腔支原体、肺炎支原体等多种支原体的检测灵敏度可达到10 CFU/ml,其中对口腔和肺炎支原体核酸拷贝数检测灵敏度可达到每毫升50基因组拷贝。结论实时荧光定量PCR法具有更高特异性和灵敏性,适用于作为质量内控方法快速检测病毒载体制品,及时发现支原体的污染,保证制品质量。Objective To investigate the feasibility and influencing factors of the two mycoplasma detection methods in viral-vectored products.Methods Two different mycoplasma nucleic acid amplification detection methods based on PCR technology were used to detect mycoplasma in two recombinant Sendai virus vector vaccines respectively expressing typeⅡherpes simplex virus antigen gD and ICP27(SeV-dF/HSV-2 gD、SeV-dF/HSV-2 ICP27).The specificity and minimum detection limit of the mycoplasma detection in harvest and purified liquids from vaccine products were studied,and the mycoplasma contamination in multiple batches of harvest liquid and purified liquid was detected.Results There was no PCR inhibition to detect mycoplasma with normal PCR method in virus harvest liquid,but the inhibition was obvious in purified liquid,and M.orale and M.pneumoniae at 100 CFU/ml were not detected.The real-time quantitative PCR(qPCR)method had no such inhibition in the detection of viral harvest and purified liquids.Moreover,the sensitivity of qPCR for M.orale,M.pneumoniae and other mycoplasma reached 10 CFU/ml,and the sensitivity of the nucleic acid copy number of M.orale and M.pneumoniae reached 50 genome copies per ml.Conclusion The qPCR method has higher specificity and sensitivity,and it can be used as a quality internal control method for rapid detection of viral vector products and timely detection of mycoplasma contamination to ensure product quality.

关 键 词：支原体 核酸扩增法 重组病毒载体制品 

分 类 号：R392[医药卫生—免疫学]