utPCR: A Strategy for the Highly Specific and Absolutely Quantitative Detection of Single Molecules within Only Minutes.
utPCR: A Strategy for the Highly Specific and Absolutely Quantitative Detection of Single Molecules within Only Minutes.
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DOI：
10.3390/bios13100910
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发表时间：
2023-09-27
期刊：
影响因子：
5.4
通讯作者：
Wang, Yongming
中科院分区：
文献类型：
--
作者：
Wang, Rui;Liu, Ying;Chen, Shuaiwei;Bai, Linlin;Guo, Kaiming;Pang, Yanan;Qian, Feng;Li, Yongfang;Ding, Li;Wang, Yongming
关键词：
血液感染是世界范围内的一个主要健康问题，死亡率极高。由于血液中的细菌浓度极低，在早期阶段检测感染是具有挑战性的。数字PCR提供了无与伦比的灵敏度，可以实现绝对定量，但耗时。此外，阴性对照中不可避免的背景信号的存在对单分子检测提出了重大挑战。在这里，我们提出了一种新的策略，称为“超快柔性薄管液滴数字PCR （utPCR）”，可以将数字PCR过程从2小时缩短到5分钟，引物退火/延长时间从几分钟缩短到5秒。重要的是，超快速PCR消除了非特异性扩增，从而实现了单分子检测。在大肠杆菌K12细胞过量106倍的高背景下，utPCR能够对大肠杆菌O157进行灵敏的检测和数字定量。此外，该方法还显示出检测血液样本中罕见病原体的潜力，检测限（LOD）可低至每mL血液10 CFU而无假阳性结果。超快（<5分钟）和高灵敏度（单分子检测），在下一代分子诊断中具有良好的前景。
Bloodstream infection is a major health problem worldwide, with extremely high mortality. Detecting infection in the early stage is challenging due to the extremely low concentration of bacteria in the blood. Digital PCR provides unparalleled sensitivity and can achieve absolute quantification, but it is time-consuming. Moreover, the presence of unavoidable background signals in negative controls poses a significant challenge for single-molecule detection. Here, we propose a novel strategy called “Ultrafast flexible thin tube-based droplet digital PCR (utPCR)” that can shorten the digital PCR process from 2 h to only 5 min, with primer annealing/extension time reduced from minutes to only 5 s. Importantly, the ultrafast PCR eliminates nonspecific amplification and thus enables single-molecule detection. The utPCR enabled the sensitive detection and digital quantification of E. coli O157 in the high background of a 106-fold excess of E. coli K12 cells. Moreover, this method also displayed the potential to detect rare pathogens in blood samples, and the limit of detection (LOD) could be as low as 10 CFU per mL of blood without false positive results. Considered ultrafast (<5 min) and highly sensitive (single-molecule detection), the utPCR holds excellent prospects in the next generation of molecular diagnosis.
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影响因子：
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