Development Of Detection Method For B.melitensis 16M Based On Bio-barcode Assay And Quantum Dots Technology

Objective:Members of the Brucella spp.are facultative intracellular bacteria that can cause global brucellosis,a zoonotic disease.Its spread and epidemic will cause huge losses to social economy and animal husbandry,as well as great harm to human health and public health.It is of great significance for its monitoring and prevention to strengthen the rapid detection of Brucella in food and products.However,the traditional detection methods have the disadvantage of high exposure risk,complex execution procedures,long operation cycle,low sensitivity and poor specificity.This study intends to overcome these shortcomings and establish a rapid,sensitive and specific detection method of Brucella based on bio-barcode assay and quantum dots technology.Methods:In this study,a method based on peptide-mediated magnetic separation?PMS?technology and Au nanoparticles?AuNPs?-mediated bio-barcode assay and quantum dots?QDs?technology is developed to achieve enrichment and purification,signal amplification and conversion and fluorescence quantitative for the rapid detection of B.melitensis 16M.Immuno-magnetic beads?IMBs?are prepared by cross-linking between streptavidin and biotin,and magnetic beads?MBs?and IMBs are characterized by transmission electron microscopy?TEM?;First,the functional AuNP-IgY bioconjugates are obtained by electrostatic adsorption and amide bonding between AuNPs and IgY,and then IgY-AuNP-probe1 are prepared by Au-S bond between the functional AuNP-IgY bioconjugates and oligonucleotide chains,and AuNPs and IgY-AuNP-probe1 are characterized by TEM and UV spectrum;First,quantum dots?QDs?are activated by cross-linking between EDC/NHS,and then QDs-probe2 are prepared by covalently binding between activated quantum dots with complementary oligonucleotide chains of 5'terminal modified amino groups,and QDs and QDs-probe2 are characterized by TEM and Fourier transform infrared spectroscopy?FTIR?.The principle of the reaction system is to enrich and isolate pathogenic bacteria in food by using IMBs,then identify the enriched pathogenic bacteria by using IgY-AuNP-probe1,realize the conversion and amplification of detection signals,and finally use fluorescent QDs-probe2 combined with IgY-AuNP-probe1 to realize the secondary conversion of signals.Results:In the characterization of nanoprobes,the low-resolution TEM morphology of MBs and successfully obtained IMBs all showes spherical and relatively good dispersion,with an average particle size of 346.71±17.03 nm and 347.25±11.44 nm,respectively.Compared with magnetic nanoparticles,the outermost surface of the IMBs has a layer of more transparent clouded film layer under high-resolution TEM,and 3%skimmed milk powder is used as best blocking buffer for IMBs;The successfully synthesized AuNPs exhibit a monodisperse uniform spherical shape under TEM with an average particle size of 17.36±1.95 nm,and the characteristic absorption peak of the UV spectrum of AuNPs is 520 nm.The double labeled IgY-AuNP-probe1 characteristic absorption peak is 526 nm,a 6 nm redshift occurs,the best PH value of AuNPs solution is 8.0,and the best dosage of IgY is 50?g.The best molar ratio of DNA/AuNP is 200:3;The TEM morphology of QDs presents a circular structure with a good dispersion,with an average particle size of 7.23±1.30nm.The FTIR of QDs has a telescopic vibration absorption peak of C=O at 1575.84cm^-1.The FTIR of QDs-probe2 shows a characteristic variation and a slight retractable vibration absorption peak at 1618.28 cm^-1 and 813.96 cm^-1,respectively.Using the prepared three bio-probes,the optimum reaction conditions of the established method system for B.melitensis 16M are as follows:The optimum concentration of IMBs is 0.2 mg/mL,and the optimum incubation time for the formation of IMB-B.melitensis 16M bioconjugates is 60 min.The final concentration and incubation time of IgY-AuNP-probe1 are 1.50 nM and 45 min,respectively.The optimal final concentration and incubation time of QD-probe2 are 2.0?g/mL and 45min,respectively.This method can detect B.melitensis 16M concentration in the range of 10 to 10⁶ cfu/mL without pre-enrichment,there's a good linear relationship,and the linear equation is I_620nm=0.7081 Log C+0.6513?R²=0.9941?,and the limit of detection?LOD?is 5.13 cfu/mL.The proposed method for the detection of B.melitensis 16M has a LOD of 1.07×10² cfu/mL and a linear range from 10² to 10⁷cfu/mL with the linear equation of I_620nm=0.4886LogC+0.1619?R²=0.9851?in milk sample,there's a good linear relationship,and a LOD of 1.72×10² cfu/ml,and a linear range from 10² to 10⁶ cfu/ml with the linear equation of I_620nm=0.7782LogC-0.2004?R²=0.9669?in lamb leach sample,there's a good linear relationship.Result of selective test shows that the method could identify B.melitensis 16M from its standard solution and its mixture with non-target bacteria,but could not recognize E.coli O157:H7,L.monocytogenes,S.aureus,S.typhimurium,and has good specificity.In short,this method takes less than 3 h to perform,and it has a low LOD and a wide linear range when testing spiked samples.Conclusion:A rapid and sensitive method for the detection of B.melitensis 16M has been developed.It is based on peptide-mediated magnetic separation technology,AuNPs-mediated bio-barcode assay technology and quantum dots technology to achieve the purpose of enrichment,separation and purification of target bacteria,signal amplification and conversion and fluorescence quantitative detection.The method has low LOD?5.13 cfu/mL?,wide linear range?10-10⁶ cfu/mL?,high sensitivity and specificity,and detection time is less than 3 hours.There are also low LOD in milk sample and lamb leach sample with 1.07×10² cfu/mL and 1.72×10²cfu/mL,respectively,and the linear range was 10²-10⁷ cfu/mL and 10²-10⁶ cfu/mL,respectively.This method can also be extended to other pathogens for rapid detection.