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<p>Pathogens pose significant threats to biosecurity and environmental health due to their potential for widespread outbreaks. Effective pathogen detection requires methods that are rapid, sensitive, specific, and informative. Here, we proposed a multiplex visual detection system that integrated ultrafast polymerase chain reaction (PCR) and molecular beacons, allowing the simultaneous detection of three pathogens in a one-pot reaction. The ultrafast PCR protocol employed cycles of just 7&#xa0;s each, allowing the entire process-from sampling to result-to be completed within only 10&#xa0;min. Molecular beacons hybridized with target sequences during ultrafast PCR, generating fluorescence signals that are visually detectable without specialized equipment. Additionally, we developed a compact, portable cartridge integrated with online software for fluorescence visualization and direct result output, eliminating the need for bulky instruments and specialized personnel, thereby facilitating point-of-care testing (POCT). The method demonstrated high specificity and sensitivity, with a limit of detection (LOD) as low as 23 copies per reaction. It achieved a 100% positive detection rate in practical applications, performing comparably to standard PCR. Furthermore, the method effectively identified low concentrations of pathogens in animal infection samples. This ultrafast, highly sensitive, specific, and informative method shows significant potential for POCT applications, including food safety monitoring and clinical diagnostics.</p>

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MVPCR: Multiplex Visual Detection Strategy Based on Ultrafast PCR for Point-of-Care Pathogens Detection Within 10 Min

  • 期刊论文

  • 发表日期：

    2025年3月4日

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  • Ziqin Zhang,
  • Cui Wu,
  • Linlin Bai,
  • Kaiming Guo,
  • Ting Wang,
  • Dianwei Liu,
  • Mingrui Zhu,
  • Feng Qian,
  • Fang Zhang,
  • Rui Wang

摘要

Pathogens pose significant threats to biosecurity and environmental health due to their potential for widespread outbreaks. Effective pathogen detection requires methods that are rapid, sensitive, specific, and informative. Here, we proposed a multiplex visual detection system that integrated ultrafast polymerase chain reaction (PCR) and molecular beacons, allowing the simultaneous detection of three pathogens in a one-pot reaction. The ultrafast PCR protocol employed cycles of just 7 s each, allowing the entire process-from sampling to result-to be completed within only 10 min. Molecular beacons hybridized with target sequences during ultrafast PCR, generating fluorescence signals that are visually detectable without specialized equipment. Additionally, we developed a compact, portable cartridge integrated with online software for fluorescence visualization and direct result output, eliminating the need for bulky instruments and specialized personnel, thereby facilitating point-of-care testing (POCT). The method demonstrated high specificity and sensitivity, with a limit of detection (LOD) as low as 23 copies per reaction. It achieved a 100% positive detection rate in practical applications, performing comparably to standard PCR. Furthermore, the method effectively identified low concentrations of pathogens in animal infection samples. This ultrafast, highly sensitive, specific, and informative method shows significant potential for POCT applications, including food safety monitoring and clinical diagnostics.