Development and validation of universal PCR, basic ERA, and qPCR assays targeting the 16S-23S rRNA intergenic spacer region for Mycoplasma detection
文献类型： 外文期刊
作者： Yin, Ruiru 1 ; Gan, Lanxi 2 ; Li, Shiyang 2 ; Zhao, Han 4 ; Wei, Yanna 2 ; Wang, Jia 2 ; Liu, Beibei 2 ; Ning, Lihua 4 ; Feng, Zhixin 1 ; Yu, Yanfei 1 ;
作者机构： 1.Nanjing Agr Univ, Coll Vet Med, Nanjing 210095, Peoples R China
2.Jiangsu Acad Agr Sci, Inst Vet Med, Key Lab Vet Biol Engn & Technol, Minist Agr & Rural Affairs, Nanjing 210014, Peoples R China
3.Hunan Agr Univ, Coll Vet Med, Changsha 410125, Peoples R China
4.Jiangsu Acad Agr Sci, Inst Germplasm Resources & Biotechnol, Nanjing 210014, Peoples R China
5.Jiangsu Univ, Sch Food & Biol Engn, Zhenjiang, Peoples R China
关键词： Mycoplasma contamination; PCR; ERA; qPCR
期刊名称：MICROBIAL PATHOGENESIS （ 影响因子：3.5； 五年影响因子：3.6 ）
ISSN： 0882-4010
年卷期： 2025 年 205 卷
页码：
收录情况： SCI
摘要： Mycoplasma contamination poses a persistent challenge in cell culture systems and the production of cell-derived biological products, including vaccines and therapeutic drugs. Current detection methods suffer from several limitations: they risk false-negative results due to incomplete species coverage, exhibit high detection limits in molecular assays, and prove time-consuming while lacking sensitivity for certain fastidious mycoplasma species that grow poorly in vitro, as outlined in pharmacopeial testing standards. To address these issues, we developed three improved detection methods-PCR, enzymatic recombinase amplification (ERA), and quantitative PCR (qPCR)-using universal primer pairs targeting conserved regions across 143 mycoplasma species. All three methods demonstrated exceptional specificity, accurately identifying 16 different Mycoplasma species (including those specified in the European Pharmacopoeia [2021] and Japanese Pharmacopoeia [JP16]) while showing no cross-reactivity with common cell culture contaminants (bacteria, viruses, or fungi). Sensitivity testing using a Spiroplasma 16S-23S spacer fragment plasmid revealed detection limits of 101 copies for PCR, 100 copies for ERA, and an impressive 10_ 1 copies for qPCR. Validation studies showed 100 % agreement with pharmacopeial gold-standard methods, with our novel methods achieving 17 % (PCR), 36.8 % (ERA), and 40.6 % (qPCR) higher detection rates than conventional approaches and outperforming comparable commercial products. Notably, all three methods significantly reduced testing time to just 1-2 h. Our results demonstrate substantial improvements in specificity, sensitivity, and detectable species range compared to existing methods. Among these approaches, qPCR emerges as particularly promising due to its superior sensitivity and rapid turnaround, making it ideally suited for quality control in biological product manufacturing and research applications. This advancement represents a significant step forward in mycoplasma contamination monitoring for the biotechnology and pharmaceutical industries.
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