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 informativ...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.展开更多
Rapid and reliable nucleic acid detection methods are essential in clinical diagnostics and biotechnology.The clustered regularly interspaced short palindromic repeats(CRISPR)system is emerging as a next-generation nu...Rapid and reliable nucleic acid detection methods are essential in clinical diagnostics and biotechnology.The clustered regularly interspaced short palindromic repeats(CRISPR)system is emerging as a next-generation nucleic acid detection technology,offering versatility,convenience and rapid detection.However,CRISPR methods are significantly limited by the protospacer adjacent motif(PAM)sequence,and achieving a one-pot reaction for detecting single nucleotide variations(SNVs)within a short time still remains challenging.Here,we developed a comprehensive method for screening PAM sequences,which significantly expands the CRISPR detection scope.Additionally,we also proposed a one-pot CRISPR method,termed"SIMPLE",capable of identifying SNVs within 30 min.We applied the SIMPLE method to the clinical diagnostics of drug-resistant bacteria and the screening of cancer hotspot mutations.The SIMPLE method successfully detected drug-resistant bacteria mediated by canonical PAM TTN sequence with a sensitivity of 10 copies per reaction and achieved 100%consistency with next-generation sequencing results.Furthermore,the SIMPLE method proved effective in detecting hotspot mutations in cancer,even at a low mutation rate of 1%in the presence of high background interference mediated by non-canonical PAM ATN sequence.Therefore,the SIMPLE method not only expands the CRISPR detection scope but also offers a one-pot reaction with high specificity for SNVs identification,making it a promising tool for next-generation molecular diagnostics.展开更多
基金the National Natural Science Foundation of China(32371521,32172285,82300220)Special Project for Experimental Animal Research(23141900300)+4 种基金the Foundation for Scholars of Fuzhou University(XRC-24068)Shanghai Rising Star Program(23QA1404300)Special Project for Medical Innovation Research(22Y11909200)Greater Bay Area Institute of Precision Medicine(Guangzhou)Human Phenome Data Center of Fudan University and Shanghai Municipal Science and Technology Major Project(2023SHZDZX02).
文摘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.
基金supported by the National Natural Science Foundation of China(32371521,82300220)Special Project for Experimental Animal Research(23141900300)+3 种基金Shanghai Rising Star Program(23QA1404300)Special Project for Medical Innovation Research(22Y11909200)Greater Bay Area Institute of Precision Medicine(Guangzhou)Human Phenome Data Center of Fudan University and Shanghai Municipal Science and Technology Major Project.(2023SHZDZX02).
文摘Rapid and reliable nucleic acid detection methods are essential in clinical diagnostics and biotechnology.The clustered regularly interspaced short palindromic repeats(CRISPR)system is emerging as a next-generation nucleic acid detection technology,offering versatility,convenience and rapid detection.However,CRISPR methods are significantly limited by the protospacer adjacent motif(PAM)sequence,and achieving a one-pot reaction for detecting single nucleotide variations(SNVs)within a short time still remains challenging.Here,we developed a comprehensive method for screening PAM sequences,which significantly expands the CRISPR detection scope.Additionally,we also proposed a one-pot CRISPR method,termed"SIMPLE",capable of identifying SNVs within 30 min.We applied the SIMPLE method to the clinical diagnostics of drug-resistant bacteria and the screening of cancer hotspot mutations.The SIMPLE method successfully detected drug-resistant bacteria mediated by canonical PAM TTN sequence with a sensitivity of 10 copies per reaction and achieved 100%consistency with next-generation sequencing results.Furthermore,the SIMPLE method proved effective in detecting hotspot mutations in cancer,even at a low mutation rate of 1%in the presence of high background interference mediated by non-canonical PAM ATN sequence.Therefore,the SIMPLE method not only expands the CRISPR detection scope but also offers a one-pot reaction with high specificity for SNVs identification,making it a promising tool for next-generation molecular diagnostics.
