Rapid Detection Of Acinetobacter Baumannii And Carbapenem-resistant Genes Based On Recombinase Polymerase Amplification

Objective: 1.To establish a real-time fluorescence detection method for rapid detection Acinetobacter baumannii(A.baumannii)based on recombinase polymerase 15 ul reaction system and amplification technique,and to detect simultaneously clinical isolates to evaluate the clinical applicability of RPA-15 ul reaction system.2.bla OXA-23 gene,the highest detection rate in the carbapenem-resistant gene of A.baumannii,was selected as the detection target gene to establish a real-time fluorescent RPA-15 ul reaction system rapidly detecting drug resistance genes.Achieved simultaneous rapid detection of A.baumannii and carbapenem resistance genes,and helped clinical rapid selection of sensitive antibiotics.Methods: 1.According to the specific conserved sequence of bla OXA-51 gene and 16s-23 s r RNA ITS gene of A.baumannii,a series of primers and probes for RPA were designed according to the design principle was provided by Twist DX Inc.After primer screening,the optimal primers and probes of bla OXA-51 gene and 16s-23 s r RNA ITS gene were selected to perform specificity and sensitivity evaluation,respectively.A.baumannii and four non-Acinetobacter of Pseudomonas aeruginosa,Candida albicans,Staphylococcus aureus,and Escherichia coli as negative control were amplified by PCR,q PCR and RPA-15 ul reaction system to detect the specificity of primer of bla OXA-51 identification gene and 16s-23 s r RNA ITS identification gene,respectively.The template was extracted after dilution of the bacterial solution,and the sensitivity of two identification genes of A.baumannii was verified by the above three amplification techniques.Furthermore,compared the sensitivity of the three methods and the sensitivity of the two identification genes were detected by RPA-15 ul reaction system.At the same time,to evaluate the clinical applicability of the RPA-15 ul reaction system,30 strains of A.baumannii clinical isolates were detected by PCR and RPA-15 ul reaction system using bla OXA-51 identification gene and 16s-23 s r RNA ITS identification gene,respectively.2.Using 30 clinical isolates of A.baumannii as the research object,the sensitivity of30 clinical isolates to 19 antibiotics was detected by the Kirby-Bauer method.According to the primers and probes design principle of RPA that was provided by Twist DX Inc,we design primers and probes for the carbapenem-resistant gene bla OXA-23 gen of A.baumannii.Then,the target DNA of 30 clinical isolates of A.baumannii were amplified respectively by PCR and RPA-15 ul reaction system,to assess the detection rate of the two detection methods and the feasibility of RPA-15 ul reaction system.Moreover,combined with antibiotic susceptibility test,we analyze the relationship between carbapenem-resistant Acinetobacter baumannii(CRAB)and bla OXA-23 gene of carbapenem-resistant gene and the feasibility of RPA-15 ul reaction system to detect the drug-resistant gene.Results: 1.The sensitivity of the RPA-15 ul reaction system to detect the bla OXA-51 identification gene and 16s-23 s r RNA ITS identification gene of A.baumannii were 2.86 CFU/ml,which were equivalent to the sensitivity of PCR and q PCR.Among the three detection methods,the specificity of the two identified genes were 100%.The detection rate that RPA-15 ul reaction system detected clinical isolates by amplifying two identified genes was 100%,which was completely consistent with PCR.The results of the Kirby-Bauer method to detect 30 clinical isolates of A.baumannii were as follows:8/30(26.7%)of strains were resistant to minocycline,and 10/30(33.3%)strains were intermediary,12/30(40.0%)showed sensitivity;17/30(56.7%)of isolates were resistant to cefoperazone/sulbactam,8/30(26.7%)showed intermediary,and 5/30(16.6%)showed sensitivity;27/30(90.0%)of isolates were resistant to amikacin,meropenem and imipenem,3/30(10.0%)strains were sensitive;28/30(93.3%)of isolates were resistant to netilmicin,gentamicin,ceftazidime and tobramycin,only 2/30(6.7%)showed sensitivity;29/30(96.7%)of isolates were resistant to ampicillin/sulbactam,ciprofloxacin,tetracycline,cefepime,cefotaxime,levofloxacin,piperacillin,piperacillin/tazobactam,only 1/30(3.3%)strains was sensitive;30/30(100%)strains were resistant to sulfamethoxazole trimethoprim;30/30(100.0%)clinical isolates of A.baumannii were all sensitive to polymyxin B.RPA-15 ul reaction system detectes the bla OXA-23 carbapenem-resistant gene of A.baumannii,showed 27/30(90%)strains appeared amplification signals,only 3/30(10%)strains were negative.Combined with antibiotic susceptibility test,3 clinical isolates that did not show amplification were sensitive to meropenem and imipenem,that is,the detection rate of bla OXA-23 resistance gene in 27 strains of CRAB was 100%,while bla OXA-23 resistance gene was not detected in carbapenem-sensitive Acinetobacter baumannii(CSAB);the results of RPA-15 ul reaction system were completely consistent with the results of PCR amplification.Conclusion: 1.This study established a real-time fluorescence RPA-15 ul reaction system method to rapid detection of A.baumannii.Both of the identification genes bla OXA-51 and 16s-23 s r RNA ITS showed high sensitivity,high specificity,rapid detection,and strong clinical applicability,RPA-15 ul reaction system is cheaper than the 50 ul reaction system of standard RPA,which is very suitable for Point-of-Care testing(POCT)and promotion in remote areas.2.Established the real-time fluorescence RPA-15 ul reaction system to rapidly detect bla OXA-23 carbapenem-resistant gene,achieved rapid detection of A.baumannii and drug resistance genes for less than 50min;we found bla OXA-23 gene of carbapenem resistance gene was closely related to CRAB,which was helpful for clinical choose rapidly sensitive antibiotics,it was great important to slow down the production of A.baumannii resistant strains.