Data Sheet 1_Application of next-generation sequencing for detecting Mycoplasma contamination in veterinary vaccines.fasta

Ensuring the safety and efficacy of veterinary vaccines requires reliable methods for detecting microbial contamination, particularly from Mycoplasma species, which pose a significant risk in cell-culture-derived vaccines. In the Republic of Korea, polymerase chain reaction (PCR) is predominantly used for Mycoplasma testing due to its faster turnaround compared to culture-based methods. However, in combination with vaccines containing Erysipelothrix rhusiopathiae and classical swine fever virus, PCR is rendered ineffective because of cross-reactivity between Mycoplasma universal primers and E. rhusiopathiae, resulting in non-specific amplification. This limitation necessitates reliance on the labor-intensive culture method, underscoring the need for more accurate and efficient alternatives. This study aimed to develop and validate next-generation sequencing (NGS)-based methods for detecting Mycoplasma contamination in veterinary vaccines and to compare their performance with that of PCR. Five species, including Acholeplasma laidlawii (genus Acholeplasma) and four Mycoplasma species—Mycoplasma fermentans, Mycoplasma orale, Mycoplasma hyorhinis, and Mycoplasma synoviae–were spiked into samples containing E. rhusiopathiae, a common vaccine component. Two NGS-based approaches were evaluated: (1) a reference-mapping method incorporating two-step alignment and de novo assembly, and (2) a 16S rRNA-based metabarcoding analysis using DADA2 and Qiime2. The reference-mapping method effectively filtered non-specific reads and accurately reconstructed Mycoplasma-derived contigs, whereas the metabarcoding approach enabled taxonomic profiling with quantitative resolution. The detection limits of NGS-based methods were substantially lower than those of PCR, demonstrating improvements of up to 100-fold depending on the species. Notably, omission of the initial mapping step resulted in excessive non-specific contig formation, highlighting the importance of the dual-step reference-mapping strategy. Although metabarcoding provided valuable abundance data, it was more prone to non-specific hits due to limited read overlap. In conclusion, the reference-mapping method demonstrated superior sensitivity, specificity, and quantification compared to both conventional PCR and metabarcoding, supporting its use as a robust tool for vaccine quality control. Implementing NGS-based detection methods could significantly enhance the safety and effectiveness of veterinary vaccines, ultimately enhancing vaccine quality control.

保障兽用疫苗的安全性与有效性，亟需可靠的微生物污染检测方法，尤其是针对支原体（Mycoplasma）类污染——这类污染在细胞培养来源的疫苗中会构成显著风险。在大韩民国，聚合酶链式反应（PCR）是支原体检测的主流手段，因其相较于培养法具有更短的检测周期。然而，当PCR用于检测含有猪红斑丹毒丝菌（Erysipelothrix rhusiopathiae）与猪瘟病毒的疫苗样本时，会因支原体通用引物与猪红斑丹毒丝菌之间的交叉反应而失效，进而引发非特异性扩增。这一局限使得检测不得不依赖耗时耗力的培养法，也凸显了开发更精准高效的替代检测手段的迫切需求。本研究旨在开发并验证基于下一代测序（NGS）的兽用疫苗支原体污染检测方法，并将其检测性能与PCR法进行对比。研究人员将5种微生物（包括无胆甾原体属的莱氏无胆甾原体（Acholeplasma laidlawii），以及4种支原体：发酵支原体（Mycoplasma fermentans）、口支原体（Mycoplasma orale）、猪鼻支原体（Mycoplasma hyorhinis）和滑膜支原体（Mycoplasma synoviae））接种至含有常见疫苗组分猪红斑丹毒丝菌的样本中。本次研究评估了两种基于NGS的检测策略：（1）结合双段比对与从头组装的参考基因组映射法；（2）依托DADA2与Qiime2工具开展的基于16S rRNA的宏条形码分析。参考基因组映射法可有效滤除非特异性测序读段，并精准重建支原体来源的重叠群（contig）；而宏条形码分析法则可完成具备定量分辨率的物种分类鉴定。基于NGS的检测方法的检出限显著低于PCR法，针对不同菌种的检测灵敏度最高可提升100倍。值得注意的是，省略初始映射步骤会导致过多非特异性重叠群生成，由此凸显了双段参考基因组映射策略的重要性。尽管宏条形码分析法可提供有价值的丰度数据，但由于测序读段重叠度有限，该方法更易出现非特异性匹配结果。综上，相较于传统PCR法与宏条形码分析法，参考基因组映射法展现出更优异的灵敏度、特异性与定量能力，可作为疫苗质量控制的可靠工具。推广基于NGS的检测方法，可显著提升兽用疫苗的安全性与有效性，最终优化疫苗质量控制流程。