基于噬菌体展示技术筛选特异性M13噬菌体构建电化学生物传感器用于流产布鲁氏菌定量检测OA

Objective Brucella abortus(B.abortus)is one of the primary causative agents of brucellosis,posing potential threats to livestock production and public health security.This study aims to screen specific M13 phages targeting the bacteria and to establish a phage-functionalized electrochemical biosensor,thereby validating its detection performance and application potential in actual sample testing.Methods Using B.abortus as a target,biopanning of a phage display peptide library was performed,combined with negative screening to enrich candidate M13 phage clones.Indirect ELISA was employed to assess the affinity and specificity of each clone.The phages were immobilized onto a gold electrode surface,with bovine serum albumin used to block non-specific sites to construct an electrochemical biosensor.Differential pulse voltammetry(DPV)current response values were employed as an indicator to optimize phage titer,phage incubation time,and sample incubation time.The DPV current response values were measured for concentrations ranging from 1.0×103 to 1.0×107 CFU/mL.A four-parameter logistic function was used to fit the standard curve,and the limit of detection(LOD)was calculated.Staphylococcus aureus,Staphylococcus epidermidis,Acinetobacter baumannii,and Escherichia coli were selected as negative controls to evaluate the specificity of the sensor.The same concentration of B.abortus samples was repeatedly detected 10 times,and the relative standard deviation(RSD)was calculated to assess repeatability.The sensor was stored at 4℃,and B.abortus was detected at different time points to calculate the RSD and evaluate the stability.B.abortus bacterial suspensions were prepared as spiked serum samples using fetal bovine serum as the matrix.SPF female BALB/c mice(6 to 8 weeks old,weighing 16 to 18 g)were randomly divided into a control group and an infected group,with 5 animals in each group.The infection group was intraperitoneally inoculated with B.abortus,and the blank group was inoculated with the same volume of PBS.Mouse spleen samples were collected.Both the constructed electrochemical biosensor and plate counting method were used to detect the above 2 types of samples.The recovery rate was calculated to evaluate the detection accuracy of the sensor in actual samples.Results Through biopanning and indirect ELISA validation,M13 phage clone P-1 capable of specifically binding to B.abortus was screened,and its binding affinity to B.abortus was superior to that of other control strains.The optimal construction conditions for the sensor were as follows:phage titer of 1.0×1011 PFU/mL,phage incubation time of 2 h,and sample incubation time of 1 h.The fitting equation for this sensor within the bacterial concentration range of 1.0×103 to 1.0×107 CFU/mL is I/μA=81.13+(184.08-81.13)/(1+Log10 C/5.01)8.68(R2=0.999 9),with an LOD of 157 CFU/mL.The sensor demonstrated favorable detection specificity(P<0.000 1)and repeatability(RSD=2.70%),with stable performance(RSD=2.37%).The recovery rate of B.abortus detected by the sensor in fetal bovine serum samples ranged from 98.2%to 106.0%,while the bacterial load in spleen samples from infected mouse model was detected as 1.23×103 CFU/mL,and the accuracy of this method was consistent with the gold standard plate culture method.Conclusion A specific M13 phage clone,P-1,targeting B.abortus was identified through screening,and this clone was used to construct an electrochemical biosensor capable of quantitatively detecting the target bacterium.