Rapid Detection Of Carbapenem-resistant Genes Of Acinetobacter Baumannii Based On LAMP

Objective:Acinetobacter baumannii is an important opportunistic pathogen associated with a variety of nosocomial infections.Carbapenems have been considered the agents of choice for the treatment of multiple drug-resistant A.baumannii.But the prevalence of carbapenem-resistant A.baumannii（CRAB）has been steadily increasing in recent years.A rapid and sensitive molecular detection in clinical isolates is quite needed for the appropriate therapy and outbreak control of A.baumannii.The loop-mediated isothermal amplification technology（LAMP）is simple,sensitive,efficient,specific,and rapid for the rapid detection of of pathogenic bacteria and drug resistance genes.This experiment aims to design the LAMP based assay for the accurate and rapid detection of A.baumannii and its resistance to carbapenem,which will have guiding significance for clinical diagnosis and treatment.Methods:1.Based on the bla_OXA-23,bla_VIM,bla _IMP,bla _NDM and bla_KPCPC gene sequences disclosed in NCBI GenBank（http://www.ncbi.nlm.nih.gov）,Primer Explorer V4 primer design software was used to design primer for these 6 target gene sequences.Select the best primers,and perform specific and sensitive experiments.2.By using the detection method of Acinetobacter baumannii established in the previous study,88 strains of initially identified Acinetobacter baumannii were re-detected,and the strains with inconsistent detection results were verified by 16S sequencing.3.For the strain identified as Acinetobacter baumannii in the previous step,the resistance gene was detected by LAMP method and PCR method respectively,and the consistency test was performed.4.The above-mentioned Acinetobacter baumannii were tested for resistance to imipenem and meropenem in vitro,and the association between resistance of the strain to imipenem and meropenem with the resistance gene was analized.Results:1.The OXA-23 primer set,VIM-1 primer set,IMP-2 primer set,NDM-3primer set,and the KPC-7 primer set showed amplification curves at the template concentrations of 10^-5,10^-4,and 10^-3 with good sensitivity and specificity.2.According to the LAMP method,84 strains of 88 strains were positive for amplification,and there were 4 strains that were inconsistent with the results given by the laboratory..We performed 16S sequencing analysis on these 4 strains.The BLAST results from the NCBI website showed that they were:Citrobacter,and green Pseudomonas,E.coli,Enterobacter cloacae.So the accuracy of the detection of Acinetobacter baumannii based on LAMP in this study was better than laboratory bacterial identification methods,and the results could be presented within a few hours.3.The kappa value obtained by the consistency test of LAMP and PCR for bla_OXA23 gene was 0.738,with good consistency.4 strains of Acinetobacter baumannii were detected to carry bla_NDM gene by LAMP method,which was consistent with the PCR results.Neither LAMP nor PCR detected strains carrying bla_IMP and VIM genes4.Bla_OXA-23XA-23 was detected in 77.4%and 66.7%of the strains by LAMP and PCR,respectively.bla_NDMDM was detected in 4.8%by both LAMP and PCR.bla _IMP and bla_VIMIM was not detected by both methods.5.A continuous calibration chi-square test was performed on 84 strains to analyze association between carrying bla_OXA23 resistance genes and resistance to imipenem and meropenem.The value was 15.7249,P<0.001。Conclusion:1.Compared with conventional culture,detection of Acinetobacter baumannii based on LAMP assay is more accurate and faster.2.In this study,a loop-mediated isothermal amplification（LAMP）assay for bla_OXA23,bla_VIM,bla_IMP,and bla_NDM genes was constructed,with good sensitivity and specificity.3.LAMP assay and PCR detection of Acinetobacter baumannii resistance genes have good consistency.LAMP method is faster and easier to observe results.4.The bla _OXA23 resistance gene is very common in Acinetobacter baumannii tested in this experiment.The bla_NDM gene is rare and no bla _IMP and bla_VIM genes are detected.5.Acinetobacter baumannii with bla_OXA-23 resistance gene may indicate that it is more likely to be resistant to carbapenem antibiotics,and has certain guiding significance for clinical drug use.