title: Development of TaqMan real-time polymerase chain reaction for the detection of the newly emerging form of carbapenem resistance gene in clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii.
abstract: OBJECTIVE
  The newly emerging form of the so-called New Delhi Metallo-beta-lactamases (NDM-1) has been reported recently from patients worldwide and broadly thought as a potential source for the major global health problem. Thus, it is important to study the epidemiology of the so-called NDM-1 harbouring bacteria to prevent its further spread and to place effective control measures. The present study describes the use of the real-time polymerase chain reaction (PCR) assay for the detection of the bla NDM-1 gene using TaqMan probes among clinical isolates.
  
  
  METHODS
  Clinical isolates of Escherichia coli (11 strains), Klebsiella pneumoniae (17 strains) and Acinetobacter baumannii (six strains) that were resistant to either of the carbapenems (meropenem or imipenem) were included in the study. The presence of carbapenemases in such strains was confirmed using the modified Hodge test. A real-time PCR assay was optimized for the detection of NDM-1 using a cloned synthetic gene fragment followed by testing of the clinical isolates. The findings were further confirmed using PCR and gene sequencing.
  
  
  RESULTS
  TaqMan probe assay displayed a good detection limit with analytical sensitivity of the assay up to 10 copies of bla NDM-1 gene per reaction. The isolates of E. coli and K. pneumoniae revealed narrow range crossing point values (Cp values) between (12-17) cycles (mean Cp value 14), indicating number of bla NDM-1 gene copies of 106-108. The wider range of Cp values (15-34) cycles with a higher mean Cp value (23.6) was observed in A. baumannii with number of bla NDM-1 gene copies of 103-108.
  
  
  CONCLUSIONS
  The study demonstrates that real-time PCR assay based on TaqMan chemistry is a useful technique for the detection of bla NDM-1 harbouring clinical isolates of E. coli, K. pneumoniae and A. baumannii. The assay has great precision in measuring the number of bla NDM-1 gene copies per specimen of DNA.
authors.affiliation: Clinical Microbiology and Infectious Diseases, Chacha Nehru Bal Chikitsalaya, (Affiliated to Maulana Azad Medical College), Govt of NCT of Delhi, Geeta Colony, Delhi - 110 031, India. microcnbc@gmail.com
authors.forename: V
authors.initials: V
authors.lastname: Manchanda
authors.forename: S
authors.initials: S
authors.lastname: Rai
authors.forename: S
authors.initials: S
authors.lastname: Gupta
authors.forename: R S
authors.initials: RS
authors.lastname: Rautela
authors.forename: R
authors.initials: R
authors.lastname: Chopra
authors.forename: D S
authors.initials: DS
authors.lastname: Rawat
authors.forename: N
authors.initials: N
authors.lastname: Verma
authors.forename: N P
authors.initials: NP
authors.lastname: Singh
authors.forename: I R
authors.initials: IR
authors.lastname: Kaur
authors.forename: P
authors.initials: P
authors.lastname: Bhalla
mesh_terms: D040981:Acinetobacter baumannii / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001431:Bacteriological Techniques / Q000379:methods*; D015780:Carbapenems / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D005798:Genes, Bacterial; D006801:Humans; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D001618:beta-Lactamases; C552864:beta-lactamase NDM-1