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.

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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