Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology

Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

A LFD-RAA-Based Rapid Detection Method for Pseudomonas syringae pv. tabaci

This study aimed to achieve rapid detection of Pseudomonas syringae pv.tabaci,the pathogen of tobacco wildfire disease.The specific primers and probes for recombinase-aided amplification(RAA)were designed with HrpZ as the target gene.RAA was then combined with the lateral flow dipstick(LFD)to establish a LFD-RAA-based rapid detection system for the pathogen.Furthermore,the detection performance of the established method was tested.The results showed that the LFDRAA method had high specificity.The amplification could be completed after 25 min of reaction at 39℃.The sensitivity of the established method reached 0.0001 ng/μL,which was superior to that of PCR detection.Moreover,the LFD-RAA method could quickly detect P.syringae pv.tabaci from tobacco leaves,demonstrating field applicability.To sum up,the LFD-RAA method established in this study can be applied in the rapid detection and early diagnosis of tobacco wildfire disease.