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

Abstract

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

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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References 35

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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

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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