Detection of plasmid-mediated colistin-resistant and carbapenem-resistant genes by multiplex PCR

Graphical abstract


Value of the protocol
Simultaneous detection of four frequent clinically relevant carbapenem-resistant genes and mcr-1 by multiplex PCR in a single reaction. Rapid, simple, and reliable for detection of frequently clinically relevant carbapenem and colistinresistant genes (mcr-1) from pure culture. Useful for laboratory application and surveillance of carbapenem-resistant and/or colistin-resistant bacteria.
Useful for detection of isolates co-carry mcr-1 and carbapenemase genes such as mcr-1 and bla NDM .

Description of protocol
Carbapenem-resistant organisms such as bla KPC , bla NDM , bla IMP , bla OXA48-like , and the emergence of the mcr-1 gene, a plasmid-mediated gene that confers colistin resistance in Enterobacteriaceae, have both been increasingly recognized worldwide. The spread of mcr-1-encoding plasmids into carbapenem-resistant Enterobacteriaceae raises concerns about the emergence of untreatable bacteria and it poses a serious threat to public health worldwide.
Many PCR techniques have been described to detect these resistant genes; however, no PCR (especially multiplex PCR) procedure has been described for detecting both mcr-1 and carbapenemresistant genes in a single reaction. This study describes a protocol to simultaneously detect mcr-1 and frequently occurring carbapenem-resistant genes (bla KPC , bla NDM , bla IMP , bla OXA48-like ) as well as to detect co-existence of mcr-1 and carbapenem-resistant genes in a single reaction from Gram-negative bacteria.
Major equipment and supplies for PCR assay PCR thermal cycler (Takara, Japan or equivalent) PCR tubes (Nest Scientific, USA or equivalent) Sterile Eppendorf style microcentrifuge tubes (Nest Scientific, USA or equivalent) distilled water was added to the lysis buffer and the DNA solutions were stored at À20 C until PCR analysis.

Multiplex PCR analysis
The multiplex PCR assay was performed in 15-ml reaction mixtures, containing 2 ml of template, 1.5 ml of deionized water, 1X JumpStart TM REDTaq1 ReadyMix TM PCR Reaction Mix (Sigma-Aldrich, USA) and 0.53 mM of each primer ( Table 1). The composition of the reagents in the multiplex PCR is shown in the Table 2. The following PCR thermal profile was used: initial activation of DNA polymerase at 95 C for 3 min; 30 cycles of denaturation at 95 C for 30 s, primer annealing at 56 C for 30 s and extension at 72 C for 45 s, and a final extension at 72 C for 5 min. The PCR products were analyzed using gel electrophoresis for 30 min on 2% agarose gels in 0.5X TBE buffer. The gels were stained with ethidium bromide and visualized under ultraviolet light (GeneGenius Bioimaging System, SynGene). The sizes of the PCR products were determined by comparison with a molecular-sized standard (GeneRuler TM 100 bp Plus DNA ladder, Thermo Fisher Scientific).

Validation
This method has advance in case of easy to use and save cost and time to simultaneously detect 4 frequently occurring carbapenemase genes and mcr-1 in a single reaction.