The outbreak of virus-induced infectious diseases poses a global public-health challenge.Nucleic acid amplification testing(NAAT)enables early detection of pandemic viruses and plays a vital role in preventing onward ...The outbreak of virus-induced infectious diseases poses a global public-health challenge.Nucleic acid amplification testing(NAAT)enables early detection of pandemic viruses and plays a vital role in preventing onward transmission.However,the requirement of skilled operators,expensive instrumentation,and biosafety laboratories has hindered the use of NAAT for screening and diagnosis of suspected patients.Here we report development of a fully automated centrifugal microfluidic system with sample-in-answer-out capability for sensitive,specific,and rapid viral nucleic acid testing.The release of nucleic acids and the subsequent reverse transcription loop-mediated isothermal amplification(RT-LAMP)were integrated into the reaction units of a microfluidic disc.The whole processing steps such as injection of reagents,fluid actuation by rotation,heating and temperature control,and detection of fluorescence signals were carried out automatically by a customized instrument.We validate the centrifugal microfluidic system using oropharyngeal swab samples spiked with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)armored RNA particles.The estimated limit of detection for armored RNA particles is 2 copies per reaction,the throughput is 21 reactions per disc,and the assay sample-to-answer time is approximately 70 min.This enclosed and automated microfluidic system efficiently avoids viral contamination of aerosol,and can be readily adapted for virus detection outside the diagnostic laboratory.展开更多
Plant pathogens cause severe losses to crop yields and economic returns in agriculture.Despite plant tissue DNA extraction of typically constituting a preliminary step in nucleic acid-based molecular diagnostics,such ...Plant pathogens cause severe losses to crop yields and economic returns in agriculture.Despite plant tissue DNA extraction of typically constituting a preliminary step in nucleic acid-based molecular diagnostics,such lab-based methods can be time-consuming and arduous to complete many samples.To mitigate these challenges,we developed an inexpensive portable DNA extraction technique that is lightweight and suitable for deployment in sampling locations,such as fields.It includes a DNA extraction device fabricated with a Steel Microneedle Array(SMA)and a simple high-efficiency DNA extraction buffer.As a result,DNA extraction times can be reduced to within~1 min,and the eluted DNA is demonstrated to be suitable for subsequent molecular biological analyses without requiring additional purification.Cross-priming amplification(CPA)technology was first established to detect Phytophthora infestans,which achieves sensitivity attainment of 10^(–7)ng/μL.The detection result can be conveniently estimated with naked-eye visual inspection using fluorescent dsDNA binding dye.CPA was demonstrated to be more feasible than PCR-based approaches and performed well in species-specific and practicability tests.This study elucidates a novel integrated pathogen detection technique coupled with SMA-Device extraction and a modified visual CPA assay to establish and verify various field-based samples infected with multiple pathogens.Altogether,the total sample-toanswer time for pathogen detection was reduced to~1.5 h,making field-based analysis affordable and achievable for farmers or extension workers inside and outside the laboratory.展开更多
基金supported by the National Natural Science Foundation of China(91959101,21904028)Chinese Academy of Sciences(YJKYYQ20180055,YJKYYQ20190068,ZDBS-LY-SLH025)the Strategic Priority Research Program of Chinese Academy of Sciences(XDB36000000)。
文摘The outbreak of virus-induced infectious diseases poses a global public-health challenge.Nucleic acid amplification testing(NAAT)enables early detection of pandemic viruses and plays a vital role in preventing onward transmission.However,the requirement of skilled operators,expensive instrumentation,and biosafety laboratories has hindered the use of NAAT for screening and diagnosis of suspected patients.Here we report development of a fully automated centrifugal microfluidic system with sample-in-answer-out capability for sensitive,specific,and rapid viral nucleic acid testing.The release of nucleic acids and the subsequent reverse transcription loop-mediated isothermal amplification(RT-LAMP)were integrated into the reaction units of a microfluidic disc.The whole processing steps such as injection of reagents,fluid actuation by rotation,heating and temperature control,and detection of fluorescence signals were carried out automatically by a customized instrument.We validate the centrifugal microfluidic system using oropharyngeal swab samples spiked with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)armored RNA particles.The estimated limit of detection for armored RNA particles is 2 copies per reaction,the throughput is 21 reactions per disc,and the assay sample-to-answer time is approximately 70 min.This enclosed and automated microfluidic system efficiently avoids viral contamination of aerosol,and can be readily adapted for virus detection outside the diagnostic laboratory.
基金supported by the National Key Research and Development Program of the Ministry of Science and Technology(2022YFD1400104)the Talent Cultivation and Development Support Program,China Agricultural University.
文摘Plant pathogens cause severe losses to crop yields and economic returns in agriculture.Despite plant tissue DNA extraction of typically constituting a preliminary step in nucleic acid-based molecular diagnostics,such lab-based methods can be time-consuming and arduous to complete many samples.To mitigate these challenges,we developed an inexpensive portable DNA extraction technique that is lightweight and suitable for deployment in sampling locations,such as fields.It includes a DNA extraction device fabricated with a Steel Microneedle Array(SMA)and a simple high-efficiency DNA extraction buffer.As a result,DNA extraction times can be reduced to within~1 min,and the eluted DNA is demonstrated to be suitable for subsequent molecular biological analyses without requiring additional purification.Cross-priming amplification(CPA)technology was first established to detect Phytophthora infestans,which achieves sensitivity attainment of 10^(–7)ng/μL.The detection result can be conveniently estimated with naked-eye visual inspection using fluorescent dsDNA binding dye.CPA was demonstrated to be more feasible than PCR-based approaches and performed well in species-specific and practicability tests.This study elucidates a novel integrated pathogen detection technique coupled with SMA-Device extraction and a modified visual CPA assay to establish and verify various field-based samples infected with multiple pathogens.Altogether,the total sample-toanswer time for pathogen detection was reduced to~1.5 h,making field-based analysis affordable and achievable for farmers or extension workers inside and outside the laboratory.
