Fei Xiao, Yuan Zengzhi, Wellner Sandra Marina, Ma Yibing, Olsen John Elmerdahl
Key Laboratory of Animal Pathogen Infection and Immunology of Fujian Province, College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou, China.
Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Front Microbiol. 2025 Jan 28;15:1487724. doi: 10.3389/fmicb.2024.1487724. eCollection 2024.
Advancements in next-generation sequencing (NGS) have significantly accelerated the development of innovative methodologies in microbiological research. In this study, we present a novel method to quantify the net survival of gene-deletion mutants within the intracellular environment. Based on standardized Illumina short-read sequencing of genomic DNA, the method eliminates the need for specific selective markers on each deletion mutant. For validation, the method was shown to accurately quantify mutants in spiked pools of mixed mutants, showing no statistically significant differences compared to the expected values based on CFU determination ( > 0.05). Further, the method was used to quantify mutants of . Gallinarum in macrophages. Six mutants and one control strain were mixed in a pool and allowed to infect HD11 cells for 2 h. The results align with prior research results, providing evidence of the feasibility of mixed mutant infections in functional gene identification. Notably, the simplicity and standardization of the method, rooted in standard whole-genome sequencing protocols, make it easily implementable across various laboratories.
下一代测序（NGS）技术的进步显著加速了微生物学研究中创新方法的发展。在本研究中，我们提出了一种新方法，用于量化细胞内环境中基因缺失突变体的净存活率。基于基因组DNA的标准化Illumina短读长测序，该方法无需在每个缺失突变体上使用特定的选择标记。为了进行验证，该方法被证明能够准确量化混合突变体加标库中的突变体，与基于菌落形成单位（CFU）测定的预期值相比，无统计学显著差异（>0.05）。此外，该方法还用于量化鸡巨噬细胞中鸡伤寒沙门氏菌的突变体。将六个突变体和一个对照菌株混合成一个库，使其感染HD11细胞2小时。结果与先前的研究结果一致，为混合突变体感染在功能基因鉴定中的可行性提供了证据。值得注意的是，该方法基于标准的全基因组测序方案，具有简单性和标准化的特点，使其易于在各个实验室实施。
