Acinetobacter 균종 검출을 위한 종-특이 PCR 기법 개발

Abstract

Background: The accurate identification of each species of Acinetobacter by biochemical identification methods is very difficult. Even by 16S rDNA PCR sequencing, many species of Acinetobacter could not be differentiated because of high sequence similarity among them. The aim of this study is to develop species-specific PCR method for identification of frequently isolated Acinetobacter in clinical specimens. Methods: The A. calcoaceticus-, A. baumannii-, A. pittii-, A. nosocomialis-, A. lwoffii-, A. ursingii- and A. bereziniae-specific PCR primers were designed based on the nucleotide sequences of rpoB gene using PrimerSelect program. The specificity of these primers were investigated with the genomic DNAs of 19 reference strains of Acinetobacter species and 15 reference strains belong to genera other than Acinetobacter. To obtain clinical test strains of various species of Acinetobacter, 293 Acinetobacter strains identified by Vitek 2 automated system were further identified to the species level by molecular method. By performing gyrB Multiplex 1 PCR for the detection of A. baumannii and A. nosocomialis developed by Higgins, we detected 210 strains of A. baumannii. The remaining 83 strains were further identified by 16s rDNA sequencing, and rpoB gene PCR-sequencing. Previously identified strains of 89 Acinetobacter clinical isolates and 293 Acinetobacter strains identified in this study were used for evaluation of Acinetobacter species-specific PCR. Results: The species composition of 293 Acinetobacter species identified in this study is as follows: 210 A. baumannii, 62 A. nosocomialis, 4 A. pittii, 1 A. calcoaceticus, 4 A. junii, 2 A. johnsonii, 3 A. lwoffii, 3 A. bereziniae, 3 A. guillouiae, and 1 A. radioresistens. The sensitivity of the A. calcoaceticus-, A. pittii-, A. nosocomialis-, A. lwoffii-, A. ursingii- and A. bereziniae-specific PCR were 100% and the sensitivity of A. baumannii-specific PCR was 98.9%. The specificity of all the PCR methods including A. calcoaceticus-, A. baumannii-, A. pittii-, A. nosocomialis-, A. lwoffii-, A. ursingii- and A. bereziniae-specific PCR were 100%. A. baumannii- and A. nosocomialis-specific PCR primers detected 10-fold lower genomic DNA than gyrB Multiplex 1 PCR primers. In addition, A. calcoaceticus- and A. pittii-specific PCR primers also detected 10-fold lower genomic DNA than gyrB Multiplex 2 PCR primers. Conclusion: These results revealed that the A. calcoaceticus-, A. baumannii-, A. pittii-, A. nosocomialis-, A. lwoffii-, A. ursingii- and A. bereziniae-specific PCR would be useful for the detection of Acinetobacter clinical isolates at the species level.