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

doi:10.3864/j.issn.0578-1752.2022.07.016

摘要/Abstract

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,从中提取全部支原体序列,经去重复后得到181条（种）支原体（包括139个已分类的单一种类和42个未分类的支原体）的16S rRNA序列,经比对后选取高变区V6到V9为检测目标区段,设计筛选出表现最佳的检测引物,建立检测支原体的通用PCR方法。选取12种常见的支原体或2种甾原体作为待检样品对该PCR方法进行检测范围验证;取6种不同动物来源的常用传代细胞和3种常见细菌进行特异性验证;选取了5种最常见的支原体和1种甾原体进行敏感性试验;并将其与经典培养法一同对17批（种）动物病毒活疫苗（分别用于6种动物）和24份8种细胞培养物样本进行对比检测以评价其实际应用效果。【结果】建立了一种通用的支原体PCR检测方法,检测引物由2条上游引物（5′-GCAAARCTATRGARAYATAGYVGAG-3′和5′-GCAAAGGCTTAGAAATAAGTTCGGAG-3′）和1条下游引物（5′- CCARCTCYCATRGTKTGACGG-3′）组成,引物配比为3﹕1﹕4,最佳退火温度为56℃。采用该PCR方法对14个较常见的支原体种类进行检测,结果均能扩增出396—413 bp大小的特异性条带,表明该方法的检测范围符合检测要求;对6种动物传代细胞和3种常见细菌进行检测,结果均未扩增出特异性条带,表明其特异性良好;灵敏度测定结果表明该PCR方法的检测下限可达20—200 CCU的活菌,对应的核酸为1.5—15.0 pg;对共计17批病毒活疫苗和24份细胞样品的检测结果与培养法检测结果基本一致,表明本研究建立的支原体PCR检测方法与培养法有很好的一致性,而且敏感性更高。【结论】建立的支原体PCR检测方法特异性强、敏感性高、重复性好、简单快速,为细胞及病毒活疫苗中可能的支原体污染提供了一种准确可靠的快速检测方法。

关键词: 细胞, 病毒活疫苗, 支原体, 16S rRNA, PCR

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.

Key words: cell, live virus vaccine, Mycoplasma, 16S rRNA, PCR

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.

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.

图/表 7

图1

图2

图3

图4

图5

图6

表1

参考文献 40

[1]	UPHOFF C C, DREXLER H G. Detection of mycoplasma contaminations. Methods in Molecular Biology (Clifton, N J), 2013, 946:1-13. doi: 10.1007/978-1-62703-128-8_1. doi: 10.1007/978-1-62703-128-8_1
[2]	MEDVEDEVA E S, BARANOVA N B, MOUZYKANTOV A A, GRIGORIEVA T Y, DAVYDOVA M N, TRUSHIN M V, CHERNOVA O A, CHERNOV V M. Adaptation of mycoplasmas to antimicrobial agents: Acholeplasma laidlawii extracellular vesicles mediate the export of ciprofloxacin and a mutant gene related to the antibiotic target. The Scientific World Journal, 2014, 2014:150615. doi: 10.1155/2014/150615. doi: 10.1155/2014/150615
[3]	TEYSSOU R, POUTIERS F, SAILLARD C, GRAU O, LAIGRET F, BOVÉ J M, BÉBÉAR C. Detection of mollicute contamination in cell cultures by 16S rDNA amplification. Molecular and Cellular Probes, 1993, 7(3):209-216. doi: 10.1006/mcpr.1993.1030. doi: 10.1006/mcpr.1993.1030
[4]	ROBINSON L B, WICHELHAUSEN R H, ROIZMAN B. Contamination of human cell cultures by pleuropneumonialike organisms. Science, 1956, 124(3232):1147-1148. doi: 10.1126/science.124.3232.1147. doi: 10.1126/science.124.3232.1147 pmid: 13380429
[5]	VOLOKHOV D V, GRAHAM L J, BRORSON K A, CHIZHIKOV V E. Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques. Molecular and Cellular Probes, 2011, 25(2/3):69-77. doi: 10.1016/j.mcp.2011.01.002. doi: 10.1016/j.mcp.2011.01.002
[6]	赵翔, 冯建平, 孟淑芳. 支原体检查的核酸检测方法及方法学验证的思考. 中国药事, 2018, 32(8):1020-1027. doi: 10.16153/j.1002-7777.2018.08.003. doi: 10.16153/j.1002-7777.2018.08.003
	ZHAO X, FENG J P, MENG S F. Considerations on Mycoplasma detection by nucleic acid detection method and the methodological validation. Chinese Pharmaceutical Affairs, 2018, 32(8):1020-1027. doi: 10.16153/j.1002-7777.2018.08.003. (in Chinese) doi: 10.16153/j.1002-7777.2018.08.003
[7]	ROTTEM S, BARILE M F. Beware of mycoplasmas. Trends in Biotechnology, 1993, 11(4):143-151. doi: 10.1016/0167-7799(93)90089-R. doi: 10.1016/0167-7799(93)90089-R
[8]	中国兽药典委员会. 中华人民共和国兽药典-二部: 2015年版. 北京: 中国农业出版社, 2016.
	Chinese Veterinary Pharmacopoeia Commission. Chinese Veterinary Pharmacopoeia of the People's Repubilic of China. Vol 2015. Beijing: Chinese Agriculture Press, 2016. (in Chinese)
[9]	国家药典委员会. 中华人民共和国药典-一部: 2020年版. 北京: 中国医药科技出版社, 2020.
	Chinese Pharmacopoeia Commission. Chinese Pharmacopoeia of the People's Repubilic of China. Vol 2020. The Medicine Science and Technology Press of China, 2020. (in Chinese)
[10]	DREXLER H G, UPHOFF C C. Mycoplasma contamination of cell cultures. Encyclopedia of Cell Technology, 2003. doi: 10.1002/0471250570.spi054. doi: 10.1002/0471250570.spi054
[11]	YOUNG L, SUNG J, STACEY G, MASTERS J R. Detection of Mycoplasma in cell cultures. Nature Protocols, 2010, 5(5):929-934. doi: 10.1038/nprot.2010.43. doi: 10.1038/nprot.2010.43
[12]	LIGASOVÁ A, VYDRŽALOVÁ M, BURIÁNOVÁ R, BRŮČKOVÁ L, VEČEŘOVÁ R, JANOŠŤÁKOVÁ A, KOBERNA K. A new sensitive method for the detection of mycoplasmas using fluorescence microscopy. Cells, 2019, 8(12):1510. doi: 10.3390/cells8121510. doi: 10.3390/cells8121510
[13]	BAI Y, GAN Y, HUA L Z, NATHUES H, YANG H, WEI Y N, WU M, SHAO G Q, FENG Z X. Application of a sIgA-ELISA method for differentiation of Mycoplasma hyopneumoniae infected from vaccinated pigs. Veterinary Microbiology, 2018, 223:86-92. doi: 10.1016/j.vetmic.2018.07.023. doi: 10.1016/j.vetmic.2018.07.023
[14]	QASEM J A, AL-MOUQATI S A, AL-ALI E M, BEN-HAJI A. Application of molecular and serological methods for rapid detection of Mycoplasma gallisepticum infection (Avian mycoplasmosis). Pakistan Journal of Biological Sciences: PJBS, 2015, 18(2):81-87. doi: 10.3923/pjbs.2015.81.87. doi: 10.3923/pjbs.2015.81.87
[15]	BEN ABDELMOUMEN MARDASSI B, BÉJAOUI A A, OUSSAEIF L, MLIK B, AMOUNA F. A recombinant antigen-based ELISA for the simultaneous differential serodiagnosis of Mycoplasma gallisepticum, Mycoplasma synoviae, and Mycoplasma meleagridis infections. Avian Diseases, 2008, 52(2):214-221. doi: 10.1637/8071-071207-Reg.1. doi: 10.1637/8071-071207-Reg.1
[16]	JOHANSSON K E, JOHANSSON I, GÖBEL U B. Evaluation of different hybridization procedures for the detection of mycoplasma contamination in cell cultures. Molecular and Cellular Probes, 1990, 4(1):33-42. doi: 10.1016/0890-8508(90)90037-Z. doi: 10.1016/0890-8508(90)90037-Z
[17]	KONG H, VOLOKHOV D V, GEORGE J, IKONOMI P, CHANDLER D, ANDERSON C, CHIZHIKOV V. Application of cell culture enrichment for improving the sensitivity of mycoplasma detection methods based on nucleic acid amplification technology (NAT). Applied Microbiology and Biotechnology, 2007, 77(1):223-232. doi: 10.1007/s00253-007-1135-1. doi: 10.1007/s00253-007-1135-1
[18]	UPHOFF C C, DREXLER H G. Detecting mycoplasma contamination in cell cultures by polymerase chain reaction. Methods in Molecular Biology (Clifton, N J), 2011, 731:93-103. doi: 10.1007/978-1-61779-080-5_8. doi: 10.1007/978-1-61779-080-5_8
[19]	英聪, 王海光, 刘灿, 沈青春, 毕丁仁, 宁宜宝. 检测污染兽用疫苗14种支原体PCR方法的建立及应用. 中国预防兽医学报, 2014, 36(1):42-45. doi: 10.3969/j.issn.1008-0589.2014.01.11. doi: 10.3969/j.issn.1008-0589.2014.01.11
	YING C, WANG H G, LIU C, SHEN Q C, BI D R, NING Y B. The establishment of the PCR assay for detection of 14 kinds of Mycoplasma contamination in veterinary vaccines. Chinese Journal of Preventive Veterinary Medicine, 2014, 36(1):42-45. doi: 10.3969/j.issn.1008-0589.2014.01.11. (in Chinese) doi: 10.3969/j.issn.1008-0589.2014.01.11
[20]	INGEBRITSON A L, GIBBS C P, TONG C, SRINIVAS G B. A PCR detection method for testing mycoplasma contamination of veterinary vaccines and biological products. Letters in Applied Microbiology, 2015, 60(2):174-180. doi: 10.1111/lam.12355. doi: 10.1111/lam.12355
[21]	MBELO S, GAY V, BLANCHARD S, ABACHIN E, FALQUE S, LECHENET J, POULET H, DE SAINT-VIS B. Development of a highly sensitive PCR/DNA chip method to detect mycoplasmas in a veterinary modified live vaccine. Biologicals, 2018, 54:22-27. doi: 10.1016/j.biologicals.2018.05.002. doi: 10.1016/j.biologicals.2018.05.002
[22]	高广仁, 万玉林, 崔治亮, 葛玉凤, 刘学荣. 兽用生物制品中支原体污染PCR检测方法的建立与应用. 黑龙江畜牧兽医, 2018(15):163-166. doi: 10.13881/j.cnki.hljxmsy.2017.12.0345. doi: 10.13881/j.cnki.hljxmsy.2017.12.0345
	GAO G R, WAN Y L, CUI Z L, GE Y F, LIU X R. Establishment and application of PCR for detection of mycoplasma contamination in veterinary biological products. Heilongjiang Animal Science and Veterinary Medicine, 2018(15):163-166. doi: 10.13881/j.cnki.hljxmsy.2017.12.0345. (in Chinese) doi: 10.13881/j.cnki.hljxmsy.2017.12.0345
[23]	ELDERING J A, FELTEN C, VEILLEUX C A, POTTS B J. Development of a PCR method for mycoplasma testing of Chinese hamster ovary cell cultures used in the manufacture of recombinant therapeutic proteins. Biologicals, 2004, 32(4):183-193. doi: 10.1016/j.biologicals.2004.08.005. doi: 10.1016/j.biologicals.2004.08.005
[24]	GRÓZNER D, SULYOK K M, KREIZINGER Z, RÓNAI Z, JÁNOSI S, TURCSÁNYI I, KÁROLYI H F, KOVÁCS Á B, KISS M J, VOLOKHOV D, GYURANECZ M. Detection of Mycoplasma anatis, M. anseris, M. cloacale and Mycoplasma sp. 1220 in waterfowl using species-specific PCR assays. PLoS ONE, 2019, 14(7):e0219071. doi: 10.1371/journal.pone.0219071. doi: 10.1371/journal.pone.0219071
[25]	WISSELINK H J, SMID B, PLATER J, RIDLEY A, ANDERSSON A M, ASPÁN A, POHJANVIRTA T, VÄHÄNIKKILÄ N, LARSEN H, HØGBERG J, COLIN A, TARDY F. A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis. BMC Veterinary Research, 2019, 15(1):86. doi: 10.1186/s12917-019-1819-7. doi: 10.1186/s12917-019-1819-7
[26]	TOTTEN A H, LEAL S M Jr, RATLIFF A E, XIAO L, CRABB D M, WAITES K B. Evaluation of the ELITe InGenius PCR platform for detection of Mycoplasma pneumoniae. Journal of Clinical Microbiology, 2019, 57(6):e00287-e00219. doi: 10.1128/JCM.00287-19. doi: 10.1128/JCM.00287-19
[27]	JEAN A, TARDY F, ALLATIF O, GROSJEAN I, BLANQUIER B, GERLIER D. Assessing mycoplasma contamination of cell cultures by qPCR using a set of universal primer pairs targeting a 1.5 kb fragment of 16S rRNA genes. PLoS ONE, 2017, 12(2):e0172358. doi: 10.1371/journal.pone.0172358. doi: 10.1371/journal.pone.0172358
[28]	BASCUÑANA C R, MATTSSON J G, BÖLSKE G, JOHANSSON K E. Characterization of the 16S rRNA genes from Mycoplasma sp. strain F38 and development of an identification system based on PCR. Journal of Bacteriology, 1994, 176(9):2577-2586. doi: 10.1128/jb.176.9.2577-2586.1994. doi: 10.1128/jb.176.9.2577-2586.1994
[29]	AMRAM E, MIKULA I, SCHNEE C, AYLING R D, NICHOLAS R A J, ROSALES R S, HARRUS S, LYSNYANSKY I. 16S rRNA gene mutations associated with decreased susceptibility to tetracycline in Mycoplasma bovis. Antimicrobial Agents and Chemotherapy, 2015, 59(2):796-802. doi: 10.1128/AAC.03876-14. doi: 10.1128/AAC.03876-14
[30]	BAKER G C, SMITH J J, COWAN D A. Review and re-analysis of domain-specific 16S primers. Journal of Microbiological Methods, 2003, 55(3):541-555. doi: 10.1016/j.mimet.2003.08.009. doi: 10.1016/j.mimet.2003.08.009
[31]	BAHRAM M, ANSLAN S, HILDEBRAND F, BORK P, TEDERSOO L. Newly designed 16S rRNA metabarcoding primers amplify diverse and novel archaeal taxa from the environment. Environmental Microbiology Reports, 2019, 11(4):487-494. doi: 10.1111/1758-2229.12684. doi: 10.1111/1758-2229.12684
[32]	代玉立, 甘林, 滕振勇, 杨静民, 祁月月, 石妞妞, 陈福如, 杨秀娟. 玉米大斑病菌和小斑病菌交配型多重PCR检测方法的建立与应用. 中国农业科学, 2020, 53(3):527-538. doi: 10.3864/j.issn.0578-1752.2020.03.006. doi: 10.3864/j.issn.0578-1752.2020.03.006
	DAI Y L, GAN L, TENG Z Y, YANG J M, QI Y Y, SHI N N, CHEN F R, YANG X J. Establishment and application of a multiple PCR method to detect mating types of Exserohilum turcicum and Bipolaris maydis. Scientia Agricultura Sinica, 2020, 53(3):527-538. doi: 10.3864/j.issn.0578-1752.2020.03.006. (in Chinese) doi: 10.3864/j.issn.0578-1752.2020.03.006
[33]	文静, 郭勇, 邱丽娟. 耐草甘膦转EPSPS/GAT大豆多重PCR检测体系的建立及应用. 中国农业科学, 2020, 53(20):4127-4136. doi: 10.3864/j.issn.0578-1752.2020.20.003. doi: 10.3864/j.issn.0578-1752.2020.20.003
	WEN J, GUO Y, QIU L J. Establishment and application of multiple PCR detection system for glyphosate-tolerant gene EPSPS/GAT in soybean. Scientia Agricultura Sinica, 2020, 53(20):4127-4136. doi: 10.3864/j.issn.0578-1752.2020.20.003. (in Chinese) doi: 10.3864/j.issn.0578-1752.2020.20.003
[34]	NIKFARJAM L, FARZANEH P. Prevention and detection of Mycoplasma contamination in cell culture. Cell Journal, 2012, 13(4):203-212.
[35]	UPHOFF C C, DREXLER H G. Eradication of Mycoplasma contaminations from cell cultures. Current Protocols in Molecular Biology, 2014, 106:28.5.1-28.512. doi: 10.1002/0471142727.mb2805s106. doi: 10.1002/0471142727.mb2805s106
[36]	UPHOFF C C, DREXLER H G. Eradication of Mycoplasma contaminations. Basic Cell Culture Protocols, 2013, 946:15-26. doi: 10.1007/978-1-62703-128-8_2. doi: 10.1007/978-1-62703-128-8_2
[37]	UPHOFF C C, DREXLER H G. Detection of mycoplasma contaminations in cell cultures by PCR analysis. Human Cell, 1999, 12(4):229-236.
[38]	KONG F, JAMES G, GORDON S, ZELYNSKI A, GILBERT G L. Species-specific PCR for identification of common contaminant mollicutes in cell culture. Applied and Environmental Microbiology, 2001, 67(7):3195-3200. doi: 10.1128/AEM.67.7.3195-3200.2001. doi: 10.1128/AEM.67.7.3195-3200.2001
[39]	钱利武, 罗京京, 王浩, 刘小琼, 王慧. 药品GMP检查中质量控制与质量保证方面存在的主要问题及建议. 中国药事, 2020, 34(1):17-21. doi: 10.16153/j.1002-7777.2020.01.003. doi: 10.16153/j.1002-7777.2020.01.003
	QIAN L W, LUO J J, WANG H, LIU X Q, WANG H. Main problems of quality control and quality assurance in drug GMP inspection and the suggestions. Chinese Pharmaceutical Affairs, 2020, 34(1):17-21. doi: 10.16153/j.1002-7777.2020.01.003. (in Chinese) doi: 10.16153/j.1002-7777.2020.01.003
[40]	谢寅, 刘晓萌, 吕建军, 孟建华, 王秀文. 瑞士GLP法令与中国GLP规范的主要差异. 中国药事, 2016, 30(4):352-354. doi: 10.16153/j.1002-7777.2016.04.008. doi: 10.16153/j.1002-7777.2016.04.008
	XIE Y, LIU X M, LÜ J J, MENG J H, WANG X W. Major differences between Swiss GLP articles and Chinese GLP. Chinese Pharmaceutical Affairs, 2016, 30(4):352-354. doi: 10.16153/j.1002-7777.2016.04.008. (in Chinese) doi: 10.16153/j.1002-7777.2016.04.008

类别 category	生物制品名称 Biological products	原材料 Materials	阳性结果数Number of positive samples
类别 category	生物制品名称 Biological products	原材料 Materials	PCR检测 PCR detection	培养法检测Cultivation^a
细胞 Cells	健康细胞8株（种） 8 strains(8 species) of healthy cell	/	0/8	0/8
细胞 Cells	待检细胞16株（8种） 16 strains(8 species) of cell samples	/	6/16	4/16（6/16）
动物用病毒活疫苗 Live vaccines for animals	鸡用病毒活疫苗6批（品种） 6 batches (species) of live vaccines for chicken	鸡胚、CEF等 Chicken embryos, CEF, etc.	1/6	0/6
	鸭用病毒活疫苗2批（品种） 2 batches (species) of live vaccines for duck	鸭胚 Duck embryos	0/2	0/2
	鹅用病毒活疫苗1批（品种） 1 batch (species) of live vaccine for geese	鸭胚 Duck embryos	0/1	0/1
	猪用病毒活疫苗5批（品种） 3 batches (species) of live vaccines for swine	Vero、PK15、Marc145、ST等 Vero, PK15, Marc145, ST, etc.	0/5	0/5
	犬用病毒活疫苗2批（品种） 2 batches (species) of live vaccines for dog	鸡胚、Vero、MDCK、PK15等 Chicken embryos, Vero,MDCK, PK15, etc.	0/2	0/2
	草鱼用病毒活疫苗1批（品种） 1 batch (species) of live vaccines for grass carp	草鱼胚胎细胞 Embryonic cell of grass carp	0/1	0/1