半嵌套PCR协议的优化,用于放大整个尖端蛋白区域,以识别废水中的SARS-CoV-2变体
Akihiko Hata1, Meiko Shimomaki2, Hiroya Kamoshida3
1Faculty of Engineering, Toyama Prefectural University, Imizu, Toyama, Japan. a-hata@pu-toyama.ac.jp.
Food and environmental virology
|January 31, 2026
概括
这项研究优化了在废水中测序SARS-CoV-2 (严重急性呼吸系统综合征冠状病毒2) 的新方法,使得即使在低度下,也能够检测到像BA.2.75这样的各种变体.
科学领域:
- 环境微生物学环境微生物学
- 病毒学 病毒学
- 分子生物学分子生物学
背景情况:
- 基于废水的流行病学 (WBE) 对于跟踪病原体动态至关重要.
- 目前的方法在复杂的废水矩阵中与SARS-CoV-2的基因分析作斗争.
- 标准协议的有限的特异性和敏感性阻碍了变种的识别.
研究的目的:
- 开发和优化半嵌套RT-PCR试验,用于废水中SARS-CoV-2的完整S蛋白编码区域测序.
- 为了从环境样本中对SARS-CoV-2变种进行强有力的基因特征鉴定.
- 提高在低度废水样本中的变异检测灵敏度.
主要方法:
- 开发了一种针对SARS-CoV-2 S-蛋白基因的半嵌套RT-PCR试验.
- 减少了多重复合,并使用单复合反应来提高特异性.
- 使用Flongle流细胞平台测序放大碎片.
- 利用一种新的单核酸变异模式矩阵来估计变异比例.
主要成果:
- 从废水样本中成功放大了~2000bp的碎片.
- 在30/39个样本中,每组原料中实现了超过1000个SARS-CoV-2-like读数.
- 有效检测到低RNA度 (<10副本/分析物).
- 确定了多个共同循环的变体,包括XBB系,JN.1和BA.2.75.5.
- 发现BA.2.75变种,此前在临床监测中未被检测到.
结论:
- 优化的半嵌套RT-PCR试验在废水中对SARS-CoV-2的基因分析是有效的.
- 这种方法提高了检测时间变异转移,即使在低病毒载量.
- 废水监测可以揭示临床监测中错过的变体,改善公共卫生洞察力.
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