Development And Evaluation Of Complex Probe Real-time PCR For Detection Of Bacterial Resistance Gene (bla_KPCbla_NDM-1)

Objective: To establish real-time PCR detection methods with complex probes anddevelop detection kits for KPC and NDM-1-mediated carbapenem resitstance, whichcan be fast, accurately and sensitively detect the bacterial resistance gene(blaKPC,blaNDM-1) of clinical specimens or enrichment broth cultures.Methods: Based on the technical principles of the complex probe, make use of theblaKPCand blaNDM-1sequence as the target genes and find the conserved region ofthem to design primers and probes for detecting the bacterial resistance gene(blaKPC,blaNDM-1). In order to improve the efficiency and sensitivity and obtain the best results,some various factors (including the formamide concentration, magnesiumconcentration, Taq polymerase concentration, primer concentration, quantity of probesand the proportionality of two probes, annealing temperature, annealing time, nucleicacid extraction method, sample type, applicable models) in the system of real-timefluorescence PCR were optimized. Then the kit’s accuracy, sensitivity, precision,specificity and stability indicators were evaluated.Results: The real-time PCR detection method for blaKPCcan be appliedto the Roche Light Cycler2.0, Roche480, ABI7500, Bio-Rad CFX-96and so on.Optimized conditions on Roche Light Cycler2.0were ultimately confirmed as: using20l system,3%formamide,3.5mmol/L Mg2+,1.0mmol/L dNTPs mixed solution,0.5mol/L KPC primers,0.2mol/L KPC fluorescent probe,0.4mol/L KPC quenchprobe,0.15mol/L internal control primers,0.06mol/L internal control probe,1.5U/20l system Taq polymerase,0.1U/20l system UDG enzyme,1.0×104cfu/mlinternal control template. Ampli cation parameters were2min at50and2min at93°C, the cycling parameters consisted of40cycles (5s at93;20s at55; and5s at72; followed by a final extension period of10s at37). Other real-time PCR using30l system, the proportion and amount of each composition is consistent with the20l system’s. Ampli cation parameters were2min at50and2min at94, thecycling parameters consisted of40cycles (20s at94;30s at55; and10s at72;followed by a final extension period of10s at25). The best sample extractionmethod was the extraction with extraction fluid. The optimal amount of template was2l/20l system or3l/30l system.The results of evaluation test of detection kit for bacterial resistance gene (blaKPC)show that the assay accurately detects KPC-containing strains with excellent sensitivity, accuracy and repeatability. For clinical samples, the minimum detectablebacteria number was not lower than5cfu. Comparison of detection results andsequencing results show the coincidence rate was100%in testing291clinicalsamples. Additional, test results of quality control materials meet the designrequirements and the fluorescence signal of Internal control for controls and allclinical samples was detected.The development of a real-time PCR assay to detect the presence of blaNDM-1gene using complex probe and the optimized results are similar to the detectionmethod of blaKPCgene. The results of evaluation test of the detection kits for bacterialresistance gene (blaNDM-1) show that the real-time PCR assay is an accurate,repeatable and sensitive method for the detection of NDM-1-containing strains.For clinical samples, the minimum detectable bacteria number was not lower than50cfu. Comparison of detection results and sequencing results show the coincidence ratewas100%in testing361clinical samples. Additional, test results of quality controlmaterials meet the design requirements.It can be concluded through the stability study in laboratory conditions thatthe two kits’ stabilization period was24hours at37;70days at4; atleast12months under-20; freezing and thawing no more than4times.Conclusion: The validation results of accuracy, sensitivity and repeatability indicatorsof the detection kits for bacterial resistance gene (blaKPC, blaNDM-1) have allreached the design requirements. The two detection methods can meet the clinicalrequirements and had significance in rapid, accurately and specifically detecting theKPC-containing strains and NDM-1-containing strains respectively, which canprovide an effective detection tool for clinical work, environmental monitoring andhealth and epidemic prevention.