Abstract: Objective To develop a rapid,specific assay for detection of Klebsiella pneumoniae carbapenemase(KPC) type carbapenemase gene.Methods The sequences of KPC type carbapenemase gene from 11 kinds of genotypes were aligned,and then the primer pair and probe from conserved sequences were designed.The suitable PCR condition was obtained through systematical optimization of PCR reaction and thereafter the specificity,sensitivity and reproducibility were evaluated.Meanwhile,the assay was applied to detect the isolates from the clinical specimens,and then the validity of the assay was verified through direct sequencing and drug resistance experiment.Results The KPC type carbapenemase gene was identified by real time PCR accurately and quickly.Furthermore,when other strains not containing KPC type carbapenemase gene were detected,no positive results appeared.Consequently,the detection limit for control plasmid,pure culture,and mocked specimen of Klebsiella pneumoniae producing KPC type carbapenemase gene was 10 copies/μL,10 colony forming unit(CFU)/mL,and 10² CFU/mL,respcetively.When the assay was applied directly to identify the 489 isolates,the results showed that 7 were positive to Klebsiella pneumoniae,11 positive to Escherichia coli and one positive to Citrobacter.The results were the same to the results obtained from direct sequencing assay.Conclusion Real time PCR is a rapid,reliable and easy-to-perform assay for the detection of KPC type carbapenemase gene.and could be applied to clinical diagnosis and investigation of epidemic disease.Meanwhile,the results of direct sequencing indicate that the strains carrying KPC type carbapenemase-2 gene are the main bacteria in Zhejiang region.