细胞和病毒活疫苗中支原体污染的荧光定量PCR检测方法的建立

doi:10.3864/j.issn.0578-1752.2026.10.015

中国农业科学 ›› 2026, Vol. 59 ›› Issue (10): 2276-2287.doi: 10.3864/j.issn.0578-1752.2026.10.015

细胞和病毒活疫苗中支原体污染的荧光定量PCR检测方法的建立

刘丹¹^,²(), 高建帅¹(), 钱佳豪¹, 张博源¹, 李慧彤¹^,², 丁家波¹, 熊涛²(), 沈青春¹()

¹ 中国农业科学院北京畜牧兽医研究所/农业农村部动物生物安全风险预警及防控重点实验室（北方），北京 100193
² 长江大学动物科学技术学院，湖北荆州 434025

收稿日期:2025-03-09 接受日期:2026-04-07 出版日期:2026-05-16 发布日期:2026-05-20
通信作者:
沈青春，E-mail：shenqingchun@caas.cn。
熊涛，E-mail：xiongtao@hotmail.com。
联系方式: 刘丹，E-mail：1465512558@qq.com。高建帅，E-mail：gaojianshuai2023@163.com。刘丹和高建帅为同等贡献作者。
基金资助:
国家“十三五”重点研发计划(2017YFF0208601); 中国农业科学院创新工程重点项目(CAAS-CSLPDCP-202403)

Establishment of Fluorescence Quantitative PCR Detection Method for Mycoplasma Contamination in Cells and Virus Live Vaccines

LIU Dan¹^,²(), GAO JianShuai¹(), QIAN JiaHao¹, ZHANG BoYuan¹, LI HuiTong¹^,², DING JiaBo¹, XIONG Tao²(), SHEN QingChun¹()

¹ Key Laboratory of Animal Biosafety Risk Prevention and Control (North)/China Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193
² College of Animal Science and Technology, Yangtze University, Jingzhou 434025, Hubei

Received:2025-03-09 Accepted:2026-04-07 Published:2026-05-16 Online:2026-05-20

摘要/Abstract

摘要：

【背景】在生物学研究和生物制品生产中常受支原体污染困扰，传统检测方法如经典培养法耗时长、灵敏度低，而普通PCR法易受抑制剂干扰且无法定量，难以满足疫苗生产质控及科研试验中对快速精准检测的需求，【目的】通过建立一种覆盖范围广、特异性强、灵敏度高的通用型荧光定量PCR检测方法，为细胞培养物、病毒活疫苗及生物原料中支原体污染提供高效筛查工具。【方法】首先从SILVA数据库下载包含全部细菌、古菌和真菌的16S/18S rRNA参考序列的库文件SILVA_138.1_SSURef，经去冗余处理后筛选出181种/株已分类支原体的16S rRNA全长序列。经过多序列比对确定V6—V8高变区为最佳检测靶点，通过Primer Premier 5.0设计引物及探针，最终筛选出3条引物（正向引物MF1：5′-GCAAARCTATRGARAYATAGYVGAG-3′和MF2：5′-GCAAAGGCTTAGAAATAAGTTCGGAG-3′，反向引物MR：5′-CCARCTCYCATRGTKTGACGG-3′）及1条双淬灭探针（5′-FAM-ACAGRTGGTGCATGGYTGTCGTCAGCTC-BHQ1-3′），通过优化引物探针配比、反应退火温度建立支原体荧光定量PCR检测方法。方法验证包括：（1）引物探针验证：选取与细胞的支原体污染相关的11种支原体/甾原体（如鸭支原体、牛支原体、莱维德氏甾原体等）进行qPCR检测；（2）灵敏性试验：选取3种支原体（鸡毒支原体、鸡滑液囊支原体、猪肺炎支原体），经10倍梯度稀释后进行qPCR检测并绘制标准曲线；（3）特异性试验：选取常见4种细菌（沙门氏菌、产气荚膜梭菌、大肠杆菌、布鲁氏菌）和8种动物细胞（Marc145细胞、Vero细胞、CEF细胞等）进行qPCR检测；（4）重复性试验：取浓度为1.0×10⁸—1.0×10¹ copies/μL的鸡滑液囊支原体BHQ03株进行组内与组间重复性测试；（5）实际样本试验：对24批（种）动物病毒活疫苗（包括鸡、鸭、猪和犬用活疫苗）、20份8种细胞培养物样本以及8种毒种进行qPCR、普通PCR和经典培养法平行比对，以评价其实际应用效果。【结果】建立的qPCR支原体检测方法使用了3条引物和1条探针，采用两步法扩增程序，退火温度为56 ℃。采用本方法对引起细胞污染最常见的支原体/甾原体种类进行检测，结果均为阳性；特异性试验结果良好，对常见细菌和动物细胞检测结果均为阴性；重复性试验结果显示Ct值变异系数（CV）均小于2%，表明本方法稳定性好；灵敏度试验结果表明本方法检测鸡滑液囊支原体、鸡毒支原体和猪肺炎支原体最低均可检测到1—2 copies/μL；实际样本试验结果表明本研究建立的支原体qPCR检测方法与普通PCR、培养法有很好的一致性，且敏感性更高。【结论】建立的支原体通用qPCR检测方法，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、且更加快捷的检测手段。

关键词: 支原体, 荧光定量PCR, 生物制品

Abstract:

【Background】Mycoplasma contamination poses a persistent challenge in biological research and biopharmaceutical production. Conventional detection methods, such as classical culture assays, are time-consuming and lack sensitivity, while the standard PCR is prone to inhibitor interference and incapable of quantification, failing to meet the demand for rapid and precise quality control in vaccine production or laboratory settings.【Objective】This study aimed to establish a universal, highly specific, and sensitive quantitative real-time PCR (qPCR) method for efficient screening of mycoplasma contamination in cell cultures, live viral vaccines, and biological raw materials.【Method】The SILVA_138.1_SSURef database, encompassing 16S/18S rRNA sequences of bacteria, archaea, and fungi, was utilized to extract 181 non-redundant 16S rRNA sequences from classified mycoplasma species. The hypervariable V6-V8 region was identified as the optimal target via multiple sequence alignment (MEGA 11.0). Three primers (forward primers MF1: 5′-GCAAARCTATRGARAYATAGYVGAG-3′; MF2: 5′-GCAAAGGCT TAGAAATAAGTTCGGAG-3; reverse primer MR: 5′-CCARCTCYCATRGTKTGACGG-3′) and a dual-quenched TaqMan probe (5′-FAM-ACAGRTGGTGCATGGYTGTCGTCAGCTC-BHQ1-3′) were designed using Primer Premier 5.0, with primer-probe ratios and annealing temperatures optimized to establish the qPCR assay. Validation included: (1) primer-probe specificity testing against 11 Mycoplasma/Acholeplasma species (e.g., Mycoplasma anatis, Mycoplasma bovis, Ureaplasma urealyticum); (2) sensitivity assessment via 10-fold serial dilutions (1.0×10⁸-1.0×10¹ copies/μL) of Mycoplasma synoviae BHQ03, Mycoplasma gallisepticum, and Mycoplasma hyopneumoniae, with standard curves generated; (3) specificity evaluation against four common bacteria (Salmonella, Clostridium perfringens, Escherichia coli, Brucella) and eight animal cell lines (Marc145, Vero, CEF, etc.); (4) repeatability analysis (intra- and inter-assay variability) using M. synoviae BHQ03 dilutions (1.0×10⁸-1.0×10¹ copies/μL); (5) parallel testing of 24 live viral vaccine batches (poultry, swine, and canine), 20 cell culture samples (8 types), and 8 viral seed stocks via qPCR, conventional PCR, and classical culture methods.【Result】The optimized qPCR protocol employed a two-step amplification program (56 ℃ annealing temperature). Specificity testing confirmed positive detection of all 11 Mycoplasma/ Acholeplasma strains and no cross-reactivity with non-target bacteria or cell lines. Repeatability tests showed coefficient of variation (CV) values <2% for Ct values across replicates. Sensitivity assays demonstrated limits of detection (LOD) of 1-2 copies/μL for M. synoviae, M. gallisepticum, and M. hyopneumoniae. Comparative analysis of clinical samples revealed high concordance between qPCR, conventional PCR, and culture methods, with qPCR exhibiting superior sensitivity.【Conclusion】The universal qPCR method developed in this study provided an accurate, reliable, and rapid detection tool for potential mycoplasma contamination in cell cultures and live viral vaccines.

Key words: mycoplasma, fluorescence quantitative PCR, biological products

刘丹, 高建帅, 钱佳豪, 张博源, 李慧彤, 丁家波, 熊涛, 沈青春. 细胞和病毒活疫苗中支原体污染的荧光定量PCR检测方法的建立[J]. 中国农业科学, 2026, 59(10): 2276-2287.

LIU Dan, GAO JianShuai, QIAN JiaHao, ZHANG BoYuan, LI HuiTong, DING JiaBo, XIONG Tao, SHEN QingChun. Establishment of Fluorescence Quantitative PCR Detection Method for Mycoplasma Contamination in Cells and Virus Live Vaccines[J]. Scientia Agricultura Sinica, 2026, 59(10): 2276-2287.

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支原体 Mycoplasma	引物及探针 Primer and probe	序列 Sequence(5′-3′)
猪肺炎支原体 M.hyopneumoniae	Mhp-F	CCAGAACCAAATTCCTTCGCTG
	Mhp-R	ACTGGCTGAACTTCATCTGGGCTA
	Mhp-P	FAM-AGCAGATCTTAGTCAAAGTGCCCGTG-BHQ
鸡毒支原体 M.gallisepticum	MG-F	TTGGGTTTAGGGATTGGGATT
	MG-R	CCAAGGGATTCAACCATC
	MG-P	FAM-TGATGATCCAAGAACGTGAAGAACACC-BHQ1
鸡滑液囊支原体 M.synoviae	MSD-F	TGTGTTTGCGGGCTTGTATT
	MSD-R	TGCATTAGTTCCTGCATTAGTTCAA
	MSD-P	FAM-AGAAGCCGAAGCTGGAGTAGCAGT-HBQ1

支原体 Mycoplasma	稀释倍数 Dilution (copies/μL)	Ct值1 Ct value 1	Ct值2 Ct value 2	Ct值3 Ct value 3	Ct值 Ct value (mean ± SD)	变异系数 CV (%)
鸡滑液囊支原体BHQ03株M.synoviaestrain BHQ03	1×10⁸	10.63	10.267	10.332	10.41±0.16	1.52
	1×10⁷	13.467	13.429	13.442	13.45±0.02	0.12
	1×10⁶	16.471	16.462	16.455	16.46±0.01	0.04
	1×10⁵	19.969	19.968	19.939	19.96±0.01	0.07
	1×10⁴	22.816	22.827	22.884	22.84±0.03	0.13
	1×10³	25.373	25.295	25.522	25.40±0.09	0.37
	1×10²	28.728	28.751	28.689	28.72±0.03	0.09
	1×10¹	31.645	32.376	32.628	32.55±0.12	1.29

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	qPCR	PCR	培养法 Cultivation^a
细胞 Cells	健康细胞8株（种） 8 strains(8 species) of healthy cells	/	0/8	0/8	0/8
细胞 Cells	待检细胞20株（8种） 20 strains(8 species) of cell samples	/	5/20	5/20	4/20（5/20）
动物用病毒活疫苗 Live vaccines for animals	鸡用病毒活疫苗8批（品种） 8 batches (species) of live vaccines for chickens	鸡胚、CEF等 Chicken embryos,CEF,etc.	2/8	2/8	0/8
	鸭用病毒活疫苗4批（品种） 4 batches (species) of live vaccines for ducks	鸭胚 Duck embryos	0/4	0/4	0/4
	猪用病毒活疫苗8批（品种） 8 batches (species) of live vaccines for swine	Vero,PK15,Marc145,ST,etc.	0/8	0/8	0/8
	犬用病毒活疫苗4批（品种） 4 batches (species) of live vaccines for dogs	Vero,MDCK,PK15,etc.	0/4	0/4	0/4
毒种 Virus seed	牛病毒性腹泻病毒2株（1种） 2 strains(1 species)of Bovine Viral Diarrhea Virus(BVDV)	MDBK,MDB-CX,etc.	1/2	1/2	1/2
	猪细小病毒2株（2种） 2 strains(2 species)of Porcine Parvovirus(PPV)	PK15,ST,etc.	2/2	2/2	2/2
	腺病毒2株（1种） 2 strains(1 species)of Adenovirus (AAV)	鸡胚、CEF等 Chicken embryos,CEF,etc.	1/2	1/2	1/2
	猫杯状病毒2株（2种） 2 strains(2 species)of Feline Caliciviru(FCV)	F81,CRFK,etc.	2/2	2/2	2/2

细胞和病毒活疫苗中支原体污染的荧光定量PCR检测方法的建立

Establishment of Fluorescence Quantitative PCR Detection Method for Mycoplasma Contamination in Cells and Virus Live Vaccines

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引物及探针 Primer and probe	序列 Sequence(5′-3′)
MF1	GCAAARCTATRGARAYATAGYVGAG
MF2	GCAAAGGCTTAGAAATAAGTTCGGAG
MR	CCARCTCYCATRGTKTGACGG
MyT4	FAM-ACAGRTGGTGCATGGYTGTCGTCAGCTC -BHQ1

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