Objective To establish a sensitive,simple and rapid detection method for African swine fever virus(ASFV)B646L gene.Methods A recombinase-aided amplification-lateral flow dipstick(RAA-LFD)assay was developed in this st...Objective To establish a sensitive,simple and rapid detection method for African swine fever virus(ASFV)B646L gene.Methods A recombinase-aided amplification-lateral flow dipstick(RAA-LFD)assay was developed in this study.Recombinase-aided amplification(RAA)is used to amplify template DNA,and lateral flow dipstick(LFD)is used to interpret the results after the amplification is completed.The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid.In addition,30 clinical samples were tested to evaluate the performance of the RAA assay.Results The RAA-LFD assay was completed within 15 min at 37°C,including 10 min for nucleic acid amplification and 5 minutes for LFD reading results.The detection limit of this assay was found to be 200 copies per reaction.And there was no cross-reactivity with other swine viruses.Conclusion A highly sensitive,specific,and simple RAA-LFD method was developed for the rapid detection of the ASFV.展开更多
creening of foodborne pathogens is important to prevent contaminated foods from their supply chains.n this study, a portable detection device was developed for rapid, sensitive and simple detection of viable almonella...creening of foodborne pathogens is important to prevent contaminated foods from their supply chains.n this study, a portable detection device was developed for rapid, sensitive and simple detection of viable almonella using a finger-actuated microfluidic chip and an improved recombinase aided amplification (RAA) assay. Improved propidium monoazide(PMAxx) was combined with RAA to enable this device to distinguish viable bacteria from dead ones. The modification of PMAxx into dead bacteria, the magnetic xtraction of nucleic acids from viable bacteria and the RAA detection of extracted nucleic acids were performed using the microfluidic chip on its supporting device by finger press-release operations. The fluorescent signal resulting from RAA amplification of the nucleic acids was collected using a USB camera nd analyzed using a self-developed smartphone App to quantitatively determine the bacterial concenration. This device could detect Salmonella typhimurium in spiked chicken meats from 1.3 × 10^(2) CFU/m L o 1.3 × 10^(7) CFU/m L in 2 h with a lower detection limit of 130 CFU/m L, and has shown its potential for on-site detection of foodborne pathogens.展开更多
Background:Fluorescent recombinase-aided amplification(RAA)assays are increasingly being used in the detection of a variety of pathogens and have the advantages of rapidity and simplicity and similar sensitivity and s...Background:Fluorescent recombinase-aided amplification(RAA)assays are increasingly being used in the detection of a variety of pathogens and have the advantages of rapidity and simplicity and similar sensitivity and specificity,compared with real-time PCR(qPCR)assays,but they require a complex probe design.To eliminate the addition of fluorescent probes for RAA,an EvaGreen dye-based recombinase-aided amplification(EvaGreen-RAA)assay using self-avoiding molecular recognition system(SAMRS)primers was developed.Methods:The SAMRS primers effectively avoided the production of primer dimers,thus improving the detection sensitivity,while EvaGreen dye was used to quantitatively measure the amplified products in real time.Using Staphy-lococcus aureus(SA)and Listeria monocytogenes(LM)as examples,EvaGreen-RAA with SAMRS primers was developed.As a reference and comparison,a traditional fluorescence probe RAA method and a RAA with SAMRS primers(SAMRS-RAA)for detecting SA and LM were also investigated.Serial di-lutions of recombinant plasmids were used to evaluate the sensitivity of the assays.Unenriched and enriched simulated milk samples were used to eval-uate the limits of detection(LOD)of these methods.Using high-resolution melting(HRM)was used to explore the sensitivity of the dual EvaGreen-RAA assay.Results:The sensitivity of the fluorescent RAA method for detecting SA and LM was 10 copies/μL using plasmids and the sensitivity of the SAMRS-RAA and EvaGreen-RAA for detecting SA and LM plasmids was 1 copies/μL.The LOD values of the EvaGreen-RAA for SA and LM in unenriched simulated milk samples were 100 and 50 CFU/mL,respectively,and the LOD value for both SA and LM using enriched simulated milk samples was 10 CFU/mL.EvaGreen-RAA had linear amplification in real time in the range of 1-10^(5)copies/μL of the plasmids of SA and LM.The sensitivity of the dual EvaGreen-RAA assay for SA and LM was estimated to be 10^(2)CFU/mL.Conclusion:A real-time quantitative EvaGreen-RAA method for detecting SA and LM was developed,which eliminates the need to design complex RAA probes.This dye-based RAA with SARMS primers provides a new strategy for simplifying fluorescence probe RAA and allowing the detection of multiple pathogens,which has many potential applications.展开更多
基金supported by National Key R&D Program of China[2017YFC200503]National Natural Science Foundation of China[No.42077399].
文摘Objective To establish a sensitive,simple and rapid detection method for African swine fever virus(ASFV)B646L gene.Methods A recombinase-aided amplification-lateral flow dipstick(RAA-LFD)assay was developed in this study.Recombinase-aided amplification(RAA)is used to amplify template DNA,and lateral flow dipstick(LFD)is used to interpret the results after the amplification is completed.The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid.In addition,30 clinical samples were tested to evaluate the performance of the RAA assay.Results The RAA-LFD assay was completed within 15 min at 37°C,including 10 min for nucleic acid amplification and 5 minutes for LFD reading results.The detection limit of this assay was found to be 200 copies per reaction.And there was no cross-reactivity with other swine viruses.Conclusion A highly sensitive,specific,and simple RAA-LFD method was developed for the rapid detection of the ASFV.
基金funded by National Natural Science Foundation of China (No. 32071899)Walmart Foundation (No. UA2020– 154)。
文摘creening of foodborne pathogens is important to prevent contaminated foods from their supply chains.n this study, a portable detection device was developed for rapid, sensitive and simple detection of viable almonella using a finger-actuated microfluidic chip and an improved recombinase aided amplification (RAA) assay. Improved propidium monoazide(PMAxx) was combined with RAA to enable this device to distinguish viable bacteria from dead ones. The modification of PMAxx into dead bacteria, the magnetic xtraction of nucleic acids from viable bacteria and the RAA detection of extracted nucleic acids were performed using the microfluidic chip on its supporting device by finger press-release operations. The fluorescent signal resulting from RAA amplification of the nucleic acids was collected using a USB camera nd analyzed using a self-developed smartphone App to quantitatively determine the bacterial concenration. This device could detect Salmonella typhimurium in spiked chicken meats from 1.3 × 10^(2) CFU/m L o 1.3 × 10^(7) CFU/m L in 2 h with a lower detection limit of 130 CFU/m L, and has shown its potential for on-site detection of foodborne pathogens.
基金National Key R&D Program of China,Grant/Award Number:2021YFC2301102National Natural Science Foundation of China,Grant/Award Numbers:82202593,U23A20106。
文摘Background:Fluorescent recombinase-aided amplification(RAA)assays are increasingly being used in the detection of a variety of pathogens and have the advantages of rapidity and simplicity and similar sensitivity and specificity,compared with real-time PCR(qPCR)assays,but they require a complex probe design.To eliminate the addition of fluorescent probes for RAA,an EvaGreen dye-based recombinase-aided amplification(EvaGreen-RAA)assay using self-avoiding molecular recognition system(SAMRS)primers was developed.Methods:The SAMRS primers effectively avoided the production of primer dimers,thus improving the detection sensitivity,while EvaGreen dye was used to quantitatively measure the amplified products in real time.Using Staphy-lococcus aureus(SA)and Listeria monocytogenes(LM)as examples,EvaGreen-RAA with SAMRS primers was developed.As a reference and comparison,a traditional fluorescence probe RAA method and a RAA with SAMRS primers(SAMRS-RAA)for detecting SA and LM were also investigated.Serial di-lutions of recombinant plasmids were used to evaluate the sensitivity of the assays.Unenriched and enriched simulated milk samples were used to eval-uate the limits of detection(LOD)of these methods.Using high-resolution melting(HRM)was used to explore the sensitivity of the dual EvaGreen-RAA assay.Results:The sensitivity of the fluorescent RAA method for detecting SA and LM was 10 copies/μL using plasmids and the sensitivity of the SAMRS-RAA and EvaGreen-RAA for detecting SA and LM plasmids was 1 copies/μL.The LOD values of the EvaGreen-RAA for SA and LM in unenriched simulated milk samples were 100 and 50 CFU/mL,respectively,and the LOD value for both SA and LM using enriched simulated milk samples was 10 CFU/mL.EvaGreen-RAA had linear amplification in real time in the range of 1-10^(5)copies/μL of the plasmids of SA and LM.The sensitivity of the dual EvaGreen-RAA assay for SA and LM was estimated to be 10^(2)CFU/mL.Conclusion:A real-time quantitative EvaGreen-RAA method for detecting SA and LM was developed,which eliminates the need to design complex RAA probes.This dye-based RAA with SARMS primers provides a new strategy for simplifying fluorescence probe RAA and allowing the detection of multiple pathogens,which has many potential applications.
