Qi Guihong, Hao Lijun, Gan Yutong, Xin Tianyi, Lou Qian, Xu Wenjie, Song Jingyuan
Key Lab of Chinese Medicine Resources Conservation, State Administration of Traditional Chinese Medicine of the People's Republic of China, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China.
Engineering Research Center of Chinese Medicine Resource, Ministry of Education, Beijing, China.
Front Microbiol. 2024 Feb 21;15:1323572. doi: 10.3389/fmicb.2024.1323572. eCollection 2024.
The challenge of discriminating closely related species persists, notably within clinical diagnostic laboratories for invasive aspergillosis (IA)-related species and food contamination microorganisms with toxin-producing potential. We employed Analysis of the whole-GEnome (AGE) to address the challenges of closely related species within the genus and developed a rapid detection method. First, reliable whole genome data for 77 species were downloaded from the database, and through bioinformatic analysis, specific targets for each species were identified. Subsequently, sequencing was employed to validate these specific targets. Additionally, we developed an on-site detection method targeting a specific target using a genome editing system. Our results indicate that AGE has successfully achieved reliable identification of all IA-related species (, and ) and three well-known species (, and ) within the section. and AGE have provided species-level-specific targets for 77 species within the genus . Based on these reference targets, the sequencing results targeting specific targets substantiate the efficacy of distinguishing the focal species from its closely related species. Notably, the amalgamation of room-temperature amplification and genome editing techniques demonstrates the capacity for rapid and accurate identification of genomic DNA samples at a concentration as low as 0.1 ng/μl within a concise 30-min timeframe. Importantly, this methodology circumvents the reliance on large specialized instrumentation by presenting a singular tube operational modality and allowing for visualized result assessment. These advancements aptly meet the exigencies of on-site detection requirements for the specified species, facilitating prompt diagnosis and food quality monitoring. Moreover, as an identification method based on species-specific genomic sequences, AGE shows promising potential as an effective tool for epidemiological research and species classification.
区分亲缘关系密切的物种仍然是一项挑战，尤其是在临床诊断实验室中，对于侵袭性曲霉菌病（IA）相关物种以及具有产毒素潜力的食品污染微生物而言。我们采用全基因组分析（AGE）来应对该属内亲缘关系密切的物种所带来的挑战，并开发了一种快速检测方法。首先，从数据库中下载了77个物种的可靠全基因组数据，并通过生物信息学分析确定了每个物种的特异性靶点。随后，采用测序来验证这些特异性靶点。此外，我们利用基因组编辑系统开发了一种针对特异性靶点的现场检测方法。我们的结果表明，AGE已成功实现对所有IA相关物种（ 、 和 ）以及该 组内三个知名物种（ 、 和 ）的可靠鉴定。AGE为该属内的77个物种提供了物种水平特异性靶点。基于这些参考靶点，针对特异性靶点的测序结果证实了将目标物种与其亲缘关系密切的物种区分开来的有效性。值得注意的是，室温扩增与基因组编辑技术的结合表明，在简洁的30分钟时间内，能够快速准确地鉴定低至0.1 ng/μl浓度的基因组DNA样本。重要的是，该方法通过采用单一管操作模式并允许可视化结果评估，避免了对大型专业仪器的依赖。这些进展恰当地满足了对特定物种进行现场检测的需求，有助于及时诊断和食品质量监测。此外，作为一种基于物种特异性基因组序列的鉴定方法，AGE作为流行病学研究和物种分类的有效工具显示出有前景的潜力。
