Rapid And High-throughput Detection Of Pathogenic Microorganism Based On Barcoded Magnetic Beads

To meet the needs of clinical detection of pathogenic microorganisms,ensure the safety of national public health and promote the health level of people,it is very important to develop a new rapid,multiplex and automated pathogenic microorganisms’ detection platform.Most Escherichia coli strains live harmlessly with human;however,there are several pathogenic strains that have the capacity to cause diarrhea or extra intestinal disease.Five classes of diarrheagenic Escherichia coli(DEC)are associated with these disease,including enterohaemorrhagic E.coli(EHEC),enteropathogenic E.coli(EPEC),enteroinvasive E.coli(EIEC),enterotoxigenic E.coli(ETEC),enteroaggregative E.coli(EAEC).Especially the first four pathogens are comon in our country.Traditional DEC detection methods,although sensitive enough,are often too time consuming for practical use,taking days to a week to perform.Therefore,new methods that overcome this performance limitation are required.In this study,we developed a novel EDC detection method based multiplex PCR and barcoded magnetic beads(BMBs)teahnology,which was highthroughput,high-sensitivity,high-accuracy and easy to realize automation.According to the virulence genes of DEC,we selected eight characteristic genes as the target: stx1,stx2,eae,rfbe,uid A for EHEC,LT,ST for ETEC,ipaH,uid A for EIEC,eae and uid A for EPEC.Eight pairs of oligonucleotide primers,one pairs of universal primer and corresponding probes based on the gene sequence in GeneBank,were designed with the help of Primer Premier 5.0.After the five bacteria were cultured and counted,the extracted DNA were amplified by two step Ploymerase Chain Reaction(PCR).The result showed that the cycle number of the specific amplification of the first step PCR is 12,and the optimal working concentrations of primers is 0.2 μM(uid A,ipaH,stx2,stx1),0.4 μM(eae,rfbe,ST)and 0.8 μM(LT)respectively.Microarray combined with multiplex polymerase chain reaction(mPCR)was established to construct an assay for simultaneous identification of pathogens.8-plex asymmetric PCR products,labeled with Cy3,were hybridized to their complementary probe fixed on chip.The detection sensitivity were as follows: 1.3×104 CFU/mL for EHEC,2×105 CFU/mL for ETEC,4×104 CFU/mL for EPEC,7.2×104 CFU/mL for EIEC,and 1.7 CFU/mL for E.coli.Barcoded Magnetic Beads utilize digital technology to offer unmatched decoding accuracy,precise fluorescence detection,and a virtually unlimited number of barcodes for use in multiplex tests.After Carboxyl BMBs covalently bound to amino probes,BMBs and mPCR become into a new detection system,which was high-throughput and easy to realize automation.The newly developed BMBs array appeared to be specific and sensitive,with the detection sensitivity of about 1.3×104 CFU/mL for EHEC,2×104 CFU/mL for ETEC,4×104 CFU/mL for EPEC,7.2x104 CFU/mL for EIEC,1.7 CFU/mL for E.coli.To each strain,the detection sensitivity of two mothods were similar,but the BMBs array shown higher sensitivity on most of targets.