Establishment Of A Method Of Rapid Enrichment Of Bloodstream Infection Pathogens Based On RhMBL Beads

Objective:Mannose-binding lectin(MBL)is a serum C-type lectin produced by the liver.It can bind specific polysaccharide residue on the surface of bacteria,fungi,virus and other microorganisms with the participation of Ca2+.Based on the Carbohydrate binding domain(CRD)of mannose-binding lectin,the aim is to establish a laboratory operation process that does not depend on blood culture to rapidly enrich bloodstream infection pathogens based on rhMBL proteinMethod:Native PAGE detected the structure of the target protein in the non-reducing state,sds-page detected the expression of the target protein,and Western Blot identified the expression of the target protein.The target protein was purified by using His tag protein purification kit,and the protein concentration was determined by BCA methodThe direct binding of M1/M2 protein to Candida spp.was detected by Western Blot,and the binding capacity of M1/M2 protein to Candida spp.was compared by flow cytometry.Pathogenic bacteria were enriched by M1-protein A magnetic beads(hereinafter referred to as the M1 method),and also by M2 protein coated Streptomyces avidin magnetic beads(hereinafter referred to as the M2 method).Firstly,Candida enrichment efficiencies of these two beads were compared with PBS simulation samples.Then,Candida was enriched in simulated rabbit blood,and compared with gold standard blood culture for diagnosis of bloodstream infection,the time required for identification of pathogenic bacteria was compared.Western Blot was used to detect the direct binding of M1 protein to Cryptococcus neoformans.Then,the M1 method was used to enrich Cryptococcus neoformans in PBS simulated samples and rabbit blood simulated samples.Finally,three different methods were compared.Results:With His label affinity chromatography,the M1 and M2 proteins were purified.The molecular weights of M1 protein and M2 protein were about 49KDa,32KDa respectively.The native forms of these two proteins were analyzed with native PAGE under the non-reducing condition.The results showed that M1 protein existed in the form of hexamer under the natural condition,while M2 protein demonstrated in the forms of trimer,tetramer and pentamers.Western blot showed that M1 and M2 proteins could bind to the standard and clinical strains of Candida commonly found in bloodstream infection,and the binding capacities of M1 protein were greater than M2.Therefore,we suspected that the binding capacity of MBL protein CRD region to microbial surface carbohydrate depended not only on the degree of aggregation of the CRDs,but also on the spatial structure formed by the aggregation of the CRDsIn addition,compared with standard blood culture,M1 method had a sensitivity of 90.5%,specificity of 100%,positive predictive value of 100%,and negative predictive value of 97.1%.M1 protein was commonly bound to standard and clinical strains of Cryptococcus neoformans,but the binding ability was slightly different.After enrichment by M1 method,direct identification by RT-PCR took the shortest time(4.25h),but the sensitivity of identification was only up to 10 CFU/mL.The sensitivity of the latter two methods was ?1 CFU/mL,but the time was long,especially the identification time of blood culture was 109-120 hConclusion:Ml and M2 proteins could bind to most pathogenic bacteria of bloodstream infection,but the binding capacites of M1 were greater than that of M2.Based on these results,a laboratory procedure of rapid enrichment of pathogenic bacteria of bloodstream infection by M1 method was established.Based on the above experimental results,we believe that the use of M1 method in the diagnosis of pathogen bloodstream infection has important clinical values.