Rapid Detection 4 Resistance Genes In Multidrug-resistant Acinetobacter Baumannii Using Multiplex PCR And High Resolution Melting Assay

Background and purposeThe resistance genes blaoxa-23,blaAde-B,blaint-1 and blaISCR-1 play an important role in the multidrug resistance mechanism of Acinetobacter baumannii(MDRAB).The ability of quickly and accurately detecting the presence of blaoxa-23,blaAde-B,blaint1\and blaISCR-1 in MDRAB for clinical microbiology laboratories not only can help doctors understand the resistance genes carried by the bacterium,guiding rational drug use,but also can guide the departments of hospital infection control to managethe patients infected by MDRAB carrying different resistance genes.Our objective was to develop a single-tube multiplex PCR assay that can detect these four genes simultaneously using a dedicated high resolution melting(HRM)assay reagent.MethodsWe designed two pairs of primers for each of the four genes,one of which was used for preliminary screening of the target gene,and another that was used to establish the multiplex PCR assay.All the second pairs of primers to each resistance gene and the strains carrying the resistance genes of blaoxa-23,blaAde-b,blaint-1 and blaISCR-1 were added into single-tube to ampliy the 4 genes.To ensure the sensitivity and specificity of multiplex PCR,4 multiplex PCR assays were designed.The products of 4 PCR assays were seprated by separated by agarose gel electrophoresis following high resolution melting curve analysis.Seventy nine MDRAB clinical strains isolated from a general hospital between January and December in 2014 were screened for the presence of the resistance genes of blaoxa-23,blaAde-B,blaint-1 and blaiscR-i using the multiplex PCR and high resolution melting curve analysis described above.ResultsMultiplex PCR amplification generated four products with the size 39,390,234 and 187 bp,respectively.Additionally,HRM analysis showed that the melting curves of multiplex PCR amplification products produced four peaks with the same melting temperature 79.5,82.4,90.3 and 87.1 ?,respectively,meaning that the multiplex PCR could amplify the 4 expected products.A total of 79 MDRAB clinical isolates were screened for the presence of the resistance genes of blaoxa-23,blaAde-B,blaint-1 and blaiSCR-i using the multiplex PCR with HRM assay.From the melting curves,the HRM assay identified blaoxa-23 and blaAde-B in 73/79 isolates,while blaint-1 was present one isolate,both blaint-1 and blaiscR-i were present in two isolates,and blaAde-B,blaint-1 and blaiscR-i were present in three isolates.No isolates were found to be carrying all four resistance genes.ConclusionsIn this study we had successfully developed a rapid assay for detecting the resistance genes of blaoxa-23,blaAde-B,blaint-i and blaiscR-i without the need to perform agarose gel electrophoresis.Compared with traditional phenotypic assays,thismultiplex PCR with HRM assay had a number of advantages:faster and more high-throughput,high cost-effective and less comtamiation.In the clinical isolates tested,blaoxa-23 and blaAde-B were the most common.While a small amount of strains carry blaint-i and blaiSCR-1,but these strains played a very important role in the spread of antimicrobial resistance genes.