Establishment Of A Multiplex QPCR Assay For Detection Of Five Common Pathogens In Fracture-related Infection

Background: Fracture-related infection(FRI)is one of the most common and serious challenging complications after fracture surgery.Tissue culture is the most commonly used but time-consuming method for identifying FRI pathogens in clinical.Here we aimed to use the five common pathogenic bacteria in FRI to establish a simple and fast multiplex q PCR method for clinical FRI detection.Object: We aimed to use the common pathogens in FRI to establish a simpler and faster TaqMan multiplex fluorescence quantitative PCR(MqPCR)method for clinical FRI detection.Method:(1)Through literature investigation and retrospective analysis of the results of previous confirmed FRI cases in Qinghai Provincial People’s Hospital,the common causative pathogens of FRI were identified.(2)The specific genes of five common pathogenic bacteria were identified with reference to relevant domestic and foreign literature.Primers and probes were designed according to the specific sequences of these genes,construction of standard plasmids.To establish a MqPCR method for the detection of five common pathogenic bacteria.Optimization of the experimental conditions of the method,and the specificity,sensitivity,and reproducibility of the method were verified.(3)66 cases of FRI and 24 control cases were detected by tissue culture and MqPCR method.Then the sensitivity,specificity,positive predictive value(PPV),negative predictive value(NPV),Youden Index and area under the ROC curve(AUC)of the two methods were calculated respectively.Results:(1)Through literature investigation and retrospective analysis of more than 260 confirmed FRI cases in Qinghai Provincial People’s Hospital.Staphylococcus aureus(SA),Staphylococcus epidermidis(SE),Pseudomonas aeruginosa(PA),Escherichia coli(E.coli)and Enterobacter cloacae(EC)were observed the common causative pathogens,causing about 80% of FRI.(2)With reference to the relevant domestic and foreign literature,we identified the specific genes of five common pathogenic bacteria,which are the nuc gene of S.aureus,16 S gene of S.epidermidis,exotoxin A gene of P.aeruginosa,ECs1048 gene of E.coli and Omp X gene of E.cloacaes.Five pathogens,S.aureus,S.epidermidis,P.aeruginosa,E.coli and E.cloacaes,were detected,the amplification curves appeared only in their respective positive template states,and there was no cross-reaction between different pathogens.Meanwhile,the CV values were less than 0.5% for both intra-group and inter-group replicate tests,with good reproducibility and stability.It indicated that MqPCR was successfully established for five common pathogens.(3)For 66 FRI cases,tissue culture detected 63 cases(95.5%)and MqPCR detected 56 cases(84.8%).Among the24 control cases,3 cases(12.5%)and 4 cases(16.7%)were positive by tissue culture and MqPCR,respectively.The sensitivity and specificity of MqPCR were 93.3% and66.7%,while those of tissue culture were 95.4% and 87.5%,respectively.If only cases infected with five common pathogenic bacteria were considered,the specificity of MqPCR changed from 66.7% to 90.9%,which was higher than the tissue culture(87.5%).Conclusion: In conclusion,the MqPCR method with TaqMan probes has the advantages of accurate quantification,rapidity(<5h),automation,simplicity and good reproducibility.The performance of MqPCR is comparable to the tissue culture.It is promising for a wide range of applications in the initial detection of pathogenic bacteria.