| 细胞和病毒活疫苗中支原体污染的PCR检测方法建立与应用 |
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| 引用本文： | 耿仁浩,刘博,王芳,罗玉峰,曲鸿飞,范学政,秦玉明,丁家波,许冠龙,沈青春,秦爱建.细胞和病毒活疫苗中支原体污染的PCR检测方法建立与应用[J].中国农业科学,2022,55(7):1458-1468. |
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| 作者姓名： | 耿仁浩 刘博 王芳 罗玉峰 曲鸿飞 范学政 秦玉明 丁家波 许冠龙 沈青春 秦爱建 |
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| 作者单位： | 1中国兽医药品监察所,北京 1000812扬州大学兽医学院,江苏扬州 225009 |
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| 基金项目： | 国家“十三五”重点研发计划（2017YFF0208601）； |
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| 摘 要： | 目的]在生物学研究及生物制品生产中易发生支原体污染,针对我国当前支原体检验方法在时效性和敏感性上的不足,建立一种简便快速、特异敏感的支原体检验PCR方法.方法]从SILVA数据库下载包含全部细菌、古菌和真菌的核糖体rRNA小亚基(16S/18S,SSU)参考序列的库文件SILVA_123-SSURef,从中提取全部...
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| 关 键 词： | 细胞 病毒活疫苗 支原体 16S rRNA PCR |
| 收稿时间： | 2021-02-08 |
Establishment and Application of PCR Assay for Mycoplasma Contamination in Cell Culture and Live Virus Vaccine |
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| GENG RenHao,LIU Bo,WANG Fang,LUO YuFeng,QU HongFei,FAN XueZheng,QIN YuMing,DING JiaBo,XU GuanLong,SHEN QingChun,QIN AiJian.Establishment and Application of PCR Assay for Mycoplasma Contamination in Cell Culture and Live Virus Vaccine[J].Scientia Agricultura Sinica,2022,55(7):1458-1468. |
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| Authors: | GENG RenHao LIU Bo WANG Fang LUO YuFeng QU HongFei FAN XueZheng QIN YuMing DING JiaBo XU GuanLong SHEN QingChun QIN AiJian |
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| Institution: | 1China Institute of Veterinary Drug Control, Beijing 1000812College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, Jiangsu |
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| Abstract: | 【Objective】Mycoplasma contamination is prone to occur in biological research and productions. Aimed at the shortage of time-effectiveness and sensitivity of current Pharmacopoeia detection methods, a simple, rapid, specific and sensitive PCR method for mycoplasma was established. 【Method】All the sequences of mycoplasmas were extracted from the datasets file SILVA_123_ SSURef from SILVA database, which contained aligned small (16S/18S, SSU) ribosomal RNA (rRNA) sequences for all three domains of life (Bacteria, Archaea and Eukarya). 181 16S rRNA unique sequences of Mycoplasma (including 139 classified species and 42 unclassified Mycoplasma) were obtained by deduplication of the sequences. After alignment, the hypervariable regions V6 to V9 were selected as the detection target regions, and the detection primers were designed to establish a universal PCR method for mycoplasma detection. 12 kinds of mycoplasmas or 2 kinds of acholeplasmas were selected to verify the detection range of the PCR method; 6 kinds of passage cells from different animal and 3 kinds of common bacteria were selected for specific verification; 5 kinds of mycoplasmas and a kind of acholeplasma were selected for sensitivity test. In order to evaluate the performance of the assay in clinical application, 17 batches of animal live virus vaccines ( for 6 kinds of animals) and 24 samples of 8 kinds of cell cultures were subjected to test, which were compared with the classical culture method for mycoplasmas. 【Result】A general PCR method for mycoplasma was established. The detection primers were composed of 2 upstream primers (5'-GCAAARCTATRGARAYA TAGYVGAG-3' and 5'- GCAAAGGCTTAGAAATAAGTTCGGAG-3') and a downstream primer (5'- CCARCTCYCATRGTKTGA CGG - 3'), and the mixing ratio of the primers was 3:1:4. The optimal annealing temperature was 56℃. The PCR method was used to detect 14 mycoplasma species, and the results showed that 396 bp-413 bp specific bands had been amplified, which indicated that the detection range of the PCR method satisfied the detection requirements; No specific bands were amplified from 6 kinds of animal cells and 3 kinds of common bacteria with the PCR assay. The results of sensitivity test showed that the detection limit of the PCR method was 20-200 CCU, and the corresponding nucleic acid was 1.5-15.0 pg. The detection results of 17 batches of live virus vaccines and 24 cell samples were almost consistent with those of culture method, which indicated that the PCR method established in this study was consistent with culture method well, and was more sensitive. 【Conclusion】The PCR assay established in this study was specific, sensitive, easy and fast, suitable for rapid detection of mycoplasma contamination in cells and live virus vaccine. |
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| Keywords: | cell live virus vaccine Mycoplasma 16S rRNA PCR |
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