Salmonella and Listeria monocytogenes are common pathogens in dairy and meat products,posing significant risks for foodborne diseases.Timely and accurate detection of these pathogens is critical for implementing effec...Salmonella and Listeria monocytogenes are common pathogens in dairy and meat products,posing significant risks for foodborne diseases.Timely and accurate detection of these pathogens is critical for implementing effective control measures against infections.The Recombinase Polymerase Amplification(RPA)technique has emerged as a promising tool,characterized by its short amplification cycle,operational simplicity,and low equipment re-quirements.This study presented a convenient and integrated method for the simultaneous detection of Sal-monella and Listeria monocytogenes using lysis buffer-magnetic nanoparticle-based DNA extraction coupled with dual recombinase polymerase amplification-lateral flow assay(RPA-LFA).A lysis buffer comprising guanidine hydrochloride,sodium deoxycholate,and Tris-HCl was optimized to disrupt bacterial cells,and Fe3O4@Al3+nanoparticles were synthesized for efficient nucleic acid adsorption by magnetic separation.The dual RPA-LFA system enabled specific amplification of target genes(SEEPA511_RS03120 and AX10_RS05385)at 37℃,with visual readout by LFA.This method exhibited a detection limit of 5×10^(1)CFU/mL for both pathogens in pure cultures and 5×10^(2)CFU/g in artificially contaminated dairy and meat products,with no cross-reactivity to nontarget bacteria.The entire procedure,from sample lysis to result visualization,was completed within 1 h,demonstrating its potential for point-of-care testing(POCT)in food safety monitoring.This approach integrates rapid nucleic acid extraction and isothermal amplification,offering a cost-effective and portable method for onsite pathogen detection.展开更多
The nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease and is one of the most destructive plant-parasitic nematodes worldwide.As there are no effective control measures,early and accurate det...The nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease and is one of the most destructive plant-parasitic nematodes worldwide.As there are no effective control measures,early and accurate detection is crucial to prevent disease spread.Here,we developed a droplet digital PCR(ddPCR)assay and a recombinase polymerase amplification-lateral flow assay(RPA-LFA)for detecting B.xylophilus and compared these methods with real-time quantitative PCR(qPCR).The results showed that this ddPCR assay was highly specific for B.xylophilus and could be quantified through the 5S gene copy numbers.Compared to qPCR,ddPCR is more sensitive and achieves absolute quantification.Additionally,the results of the RPA-LFA were 100%consistent with the positive qPCR results but significantly reduced the required reaction time to within 30 min.Together,establishing and combining these two assays can offer an effective method for the early detection of B.xylophilus,i.e.,the ddPCR assay represents a promising alternative method for the precise quantitative detection of B.xylophilus,while the RPA-LFA is a simple,rapid and visual method that can be used for rapid detection of B.xylophilus in the field and in resource-limited conditions.展开更多
基金supported by grants Key Laboratory of Detection and Traceability Technology of Foodborne Pathogenic Microorganisms,State Administration for Market Regulation(No.ZJ-L202503).
文摘Salmonella and Listeria monocytogenes are common pathogens in dairy and meat products,posing significant risks for foodborne diseases.Timely and accurate detection of these pathogens is critical for implementing effective control measures against infections.The Recombinase Polymerase Amplification(RPA)technique has emerged as a promising tool,characterized by its short amplification cycle,operational simplicity,and low equipment re-quirements.This study presented a convenient and integrated method for the simultaneous detection of Sal-monella and Listeria monocytogenes using lysis buffer-magnetic nanoparticle-based DNA extraction coupled with dual recombinase polymerase amplification-lateral flow assay(RPA-LFA).A lysis buffer comprising guanidine hydrochloride,sodium deoxycholate,and Tris-HCl was optimized to disrupt bacterial cells,and Fe3O4@Al3+nanoparticles were synthesized for efficient nucleic acid adsorption by magnetic separation.The dual RPA-LFA system enabled specific amplification of target genes(SEEPA511_RS03120 and AX10_RS05385)at 37℃,with visual readout by LFA.This method exhibited a detection limit of 5×10^(1)CFU/mL for both pathogens in pure cultures and 5×10^(2)CFU/g in artificially contaminated dairy and meat products,with no cross-reactivity to nontarget bacteria.The entire procedure,from sample lysis to result visualization,was completed within 1 h,demonstrating its potential for point-of-care testing(POCT)in food safety monitoring.This approach integrates rapid nucleic acid extraction and isothermal amplification,offering a cost-effective and portable method for onsite pathogen detection.
基金supported by the Science and Technology Innovation Program of Xiongan New Area(2023XAGG0065)Fundamental Research Funds for the Central Universities(QNTD202510).
文摘The nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease and is one of the most destructive plant-parasitic nematodes worldwide.As there are no effective control measures,early and accurate detection is crucial to prevent disease spread.Here,we developed a droplet digital PCR(ddPCR)assay and a recombinase polymerase amplification-lateral flow assay(RPA-LFA)for detecting B.xylophilus and compared these methods with real-time quantitative PCR(qPCR).The results showed that this ddPCR assay was highly specific for B.xylophilus and could be quantified through the 5S gene copy numbers.Compared to qPCR,ddPCR is more sensitive and achieves absolute quantification.Additionally,the results of the RPA-LFA were 100%consistent with the positive qPCR results but significantly reduced the required reaction time to within 30 min.Together,establishing and combining these two assays can offer an effective method for the early detection of B.xylophilus,i.e.,the ddPCR assay represents a promising alternative method for the precise quantitative detection of B.xylophilus,while the RPA-LFA is a simple,rapid and visual method that can be used for rapid detection of B.xylophilus in the field and in resource-limited conditions.
