1. Foodborne Microorganisms

1.1 Multiplex bacteria detection using one-pot CRIS-PR/-Cas13a-based droplet microfluidics

1.2 Rapid detection of Salmonella with Recombinase Aided Amplification

1.3 Establishment and application of a method for detecting Staphylococcus aureus using asymmetric recombinase-aided amplification combined with molecular beacons

1.4 Establishment of a rapid detection method for Salmonella using recombinase-aided amplification technology

1.5 Rapid detection of group A beta-hemolytic streptococcus using recombinase-aided amplification method

1.6 Establishment of a rapid detection method for Vibrio parahaemolyticus using CRISPR-Cas13a combined with recombinase-aided amplification

1.7 Research on rapid detection of Staphylococcus aureus using CRISPR-Cas13a assisted RAA

1.8 Rapid detection of Salmonella in soil using recombinase-mediated isothermal nucleic acid amplification method

1.9 Detection of Vibrio harveyi using recombinase-mediated isothermal amplification technology

1.10 Rapid PCR detection of pathogenic microorganisms in animal-derived foods

2. Review

2.1 Research progress on the application of recombinase isothermal amplification technology in analysis and detection

Multiplex bacteria detection using one-potCRISPR/Cas13a-based droplet microfluidics

Abstract:High-throughput detection of bacteria at low levels is critical in public health,food safety,and first response.Herein,for the first time,we present a platform based on droplet microfluidics coupling with the recombinasealded amplification(RAA)-assisted one-pot clustered regularly interspaced short palindromic repeats togetherwith CRSPR-associated proteins 13a(CRISPR/Cas13a) assay,and droplet encoding strategy for accurate andsensitive deter mination of nudeic adidsfrom various foodborne pathogens.The workflow takes fulladvantageof CRISPR/Cas13a signal amplification and droplet confinement effects,which enhancesthe detection sensitivi-ty and enables end-point quantitation.Meanwhile,by varying the color of droplets,the number of bacteriadetectedatthe same time is greatly improved.Ilt possesses the capability to simultaneously detectseven differ-ent types of foodborne pathogens.Notably,the system is also applied to real food samples with satisfactoryresults.Overall,in view of superiorities inhigh sensitivity,outst anding selectivity,and large-scale multiplexing,the one-pot CRISPR/Cas13a-based droplet microfluidic system could be expanded and universalized for identi-fying other bacteria.

Key words:Bacterla,Multiplex detection,RAA,One-pot CRSPR/Cas13a assay,Droplet microfluidics,Drapletencoding.

Rapid detection of Salmonella with Recombinase Aided Amplification

Abstract:Rapid Salmonella detection using Recombinase Aided Amplification was established.The reactioncompletes in20 min at 39 ℃ and can be performed with a portable device.Once further impraved,this methodshould be a great choice for monitaring contamination,such as foodborne Salmonella or for similar purposes.

Keywords:Salmonella,Re combinase Aided Amplification,Foodborne,Food safety.

Establishment and application of a method for detecting Staphylococcus aureus using asymmetric recombinase-aided amplification combined with molecular beacons

Abstract:Objective:To establish a rapid and isothermal detection method for pathogenic bacteria based on recombinase polymerase amplification (RAA) combined with molecular beacons.Methods:Methodology establishment.Primers and molecular beacon probes were designed according to the gene corresponding to the A protein (SPA) of Staphylococcus aureus.The optimal primer concentration ratio was determined by continuously adjusting the primer concentration ratio for asymmetric amplification.Asymmetric recombinase-aided amplification was used and hybridized with molecular beacon probes.The results were observed by agarose gel electrophoresis and fluorescence acquisition.The sensitivity of the method was detected by diluting the positive plasmid in 10-fold gradients.The specificity of the method was detected by detecting 72 strains of bacteria, including Staphylococcus aureus and other bacteria of the genus Staphylococcus, preserved in the microbiology laboratory of the Department of Laboratory Medicine, Daping Hospital in December 2016.Based on the specificity experiment, 39 other strains preserved in our laboratory in December 2016 were added for Kappa consistency test and clinical diagnostic efficacy evaluation.Results:When the concentration ratio of the restrictive primer to the non-restrictive primer was 1:20, the efficiency of producing single-stranded DNA (SSDNA) was the highest.The efficiency of asymmetric RAA amplification and hybridization with molecular beacon probes was significantly higher than that of symmetric amplification.The sensitivity of the method was 20 copies/μl.The RAA hybridization method could distinguish Staphylococcus aureus from other strains of the genus Staphylococcus.Compared with the traditional gold standard, Kappa was 0.860, indicating good consistency.After detecting 111 strains of bacteria, the sensitivity of the method was 92.5% (37/40), the specificity was 97.2% (691/71), the positive predictive value was 94.9% (37/39), the negative predictive value was 95.8% (69/72), the positive likelihood ratio was 33.04, the negative likelihood ratio was 0.077, and the Youden index was 0.897.Compared with the traditional gold standard, Kappa was 0.902, and the consistency between the two methods was good.Conclusion:By optimizing the asymmetric RAA amplification conditions and combining it with molecular beacon probes, a new method for detecting bacterial DNA using RAA hybridization technology was established, laying a foundation for the application of isothermal amplification in clinical practice.(Chinese Journal of Laboratory Medicine, 2017, 40:309-313)

Keywords:Staphylococcus aureus; Nucleic acid amplification techniques; Molecular probes

Establishment of a rapid detection method for Salmonella using recombinase-aided amplification technology

Abstract:Objective:To establish a rapid detection method for Salmonella using recombinase aided amplification (RAA).Methods:Primers and probes were designed based on the invA gene of Salmonella.The sensitivity of the RAA method was analyzed by constructing plasmids containing the target gene fragment.The specificity of the method was verified by detecting Escherichia coli and Shigella, and Salmonella positive samples were detected for verification.Results:The established method was performed at 39℃, the detection time was within 20 min, the detection limit was 102 copies/μl, and there was no cross-reaction with Escherichia coli and Shigella, indicating good specificity.Conclusion:The established RAA detection method is rapid, sensitive, specific, and easy to operate, suitable for rapid detection of Salmonella.

Keywords:Salmonella; Recombinase-aided amplification; Molecular detection