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.

Rapid Internal Control Reference Recombinase-Aided Amplification Assays for EBV and CMV Detection

Epstein-Barr virus(EBV)and cytomegalovirus(CMV),two of the most prevalent human herpesviruses,cause a wide spectrum of diseases and symptoms and are associated with serious health problem.In this study,we developed an internal control reference recombinase-aided amplification(ICR-RAA)assay for the rapid detection of EBV and CMV within 30 min.The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV,respectively,with no cross reaction with other pathogens.In comparison with those of the commercial quantitative polymerase chain reaction(qPCR),the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33%and 84.84%,respectively;the specificity was 98.75%and 100.00%,respectively;and the Kappa values were 0.930 and 0.892(P<0.05),respectively.In comparison with those of qPCR,the sensitivity of the EBV and CMV ICR-RAAs using the DNA by thermal lysis was 72.22%and 80.00%,respectively;the specificity was 100.00%。