Rapid Detection of New Delhi Metallo-β-Lactamase Gene and Variants Coding for Carbapenemases with Different Activities by Use of a PCR-Based
            <i>In Vitro</i>
            Protein Expression Method

Huang, Li; Hu, Xiumei; Zhou, Maosheng; Yang, Yilin; Qiao, Jianzhong; Wang, Dianbing; Yu, Junping; Cui, Zongqiang; Zhang, Zhiping; Zhang, Xian-En; Wei, Hongping

doi:10.1128/jcm.03363-13

Cited by 7 publications

(3 citation statements)

References 30 publications

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“…This optimized method was called “m16S_qPCR”. That a Real-Time PCR of long DNA fragments can be performed with non-degenerate primers has been independently reported to detect two bacterial non-ribosomal genes [ 20 , 21 ].…”

Section: Resultsmentioning

confidence: 99%

Assessing mycoplasma contamination of cell cultures by qPCR using a set of universal primer pairs targeting a 1.5 kb fragment of 16S rRNA genes

et al. 2017

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Mycoplasmas (a generic name for Mollicutes) are a predominant bacterial contaminant of cell culture and cell derived products including viruses. This prokaryote class is characterized by very small size and lack of a cell wall. Consequently, mycoplasmas escape ultrafiltration and visualization under routine microscopic examination, hence the ease with which cells in culture can be contaminated, with routinely more than 10% of cell lines being contaminated. Mycoplasma are a formidable threat both in fundamental research by perverting a whole range of cell properties and functions and in the pharmacological use of cells and cell derived products. Although many methods have been developed, there is still a need for a sensitive, universal assay. Here is reported the development and validation of a quantitative polymerase chain reaction (qPCR) based on the amplification of a 1.5 kb fragment covering the 16S rDNA of the Mollicute class by real-time PCR using universal U1 and U8 degenerate primers. The method includes the addition of a DNA loading probe to each sample to monitor DNA extraction and the absence of PCR inhibitors in the extracted DNA, a positive mycoplasma 16S rDNA traceable reference sample to exclude any accidental contamination of an unknown sample with this reference DNA, an analysis procedure based on the examination of the melting curve and the size of the PCR amplicon, followed by quantification of the number of 16S rDNA copies (with a lower limit of 19 copies) when relevant, and, if useful, the identification of the contaminating prokaryote by sequencing. The method was validated on a collection of mycoplasma strains and by testing over 100 samples of unknown contamination status including stocks of viruses requiring biosafety level 2, 3 or 4 containments. When compared to four established methods, the m16S_qPCR technique exhibits the highest sensitivity in detecting mycoplasma contamination.

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Section: Resultsmentioning

confidence: 99%

Assessing mycoplasma contamination of cell cultures by qPCR using a set of universal primer pairs targeting a 1.5 kb fragment of 16S rRNA genes

et al. 2017

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“…A long-fragment real-time quantitative PCR-combined in vitro protein expression (PCR-P) method has been developed for detection of bla NDM-1 . PCR-P is able to detect bla NDM-1 variants that have led to changes of function by measuring rates of degradation of imipenem (222).…”

Section: Detection Of Ndm-encoding Genesmentioning

confidence: 99%

NDM Metallo-β-Lactamases and Their Bacterial Producers in Health Care Settings

et al. 2019

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SUMMARYNew Delhi metallo-β-lactamase (NDM) is a metallo-β-lactamase able to hydrolyze almost all β-lactams. Twenty-four NDM variants have been identified in >60 species of 11 bacterial families, and several variants have enhanced carbapenemase activity.Klebsiella pneumoniaeandEscherichia coliare the predominant carriers ofblaNDM, with certain sequence types (STs) (forK. pneumoniae, ST11, ST14, ST15, or ST147; forE. coli, ST167, ST410, or ST617) being the most prevalent. NDM-positive strains have been identified worldwide, with the highest prevalence in the Indian subcontinent, the Middle East, and the Balkans. MostblaNDM-carrying plasmids belong to limited replicon types (IncX3, IncFII, or IncC). Commonly used phenotypic tests cannot specifically identify NDM. Lateral flow immunoassays specifically detect NDM, and molecular approaches remain the reference methods for detectingblaNDM. Polymyxins combined with other agents remain the mainstream options of antimicrobial treatment. Compounds able to inhibit NDM have been found, but none have been approved for clinical use. Outbreaks caused by NDM-positive strains have been reported worldwide, attributable to sources such as contaminated devices. Evidence-based guidelines on prevention and control of carbapenem-resistant Gram-negative bacteria are available, although none are specific for NDM-positive strains. NDM will remain a severe challenge in health care settings, and more studies on appropriate countermeasures are required.

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“…This assay produces results within 6 hours as compared to GenoType MTBDRplus assay (Hain Lifescience GmbH, Germany) and culture susceptibility testing, which take 8 hours and 56 days to generate results. In the recent time, several in-house qPCR assays for rapid and simultaneous detection of genes encoding Klebsiella pneumoniae carbapenemase (bla KPC ) and New Delhi metallo- β -lactamase (bla NDM ) in Gram-negative rod-shaped bacteria [ 40 – 43 ] have been introduced. Similarly, several in-house qPCR assays for rapid and simultaneous detection of bla OXA-48 , bla VIM , and bla IMP carbapenemase genes in Enterobacteriaceae have been established [ 44 – 46 ].…”

Section: Genotypic Antimicrobial Resistant Detection Methodsmentioning

confidence: 99%

Emerging Rapid Resistance Testing Methods for Clinical Microbiology Laboratories and Their Potential Impact on Patient Management

Frickmann

Masanta

Zautner

2014

BioMed Research International

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Atypical and multidrug resistance, especially ESBL and carbapenemase expressing Enterobacteriaceae, is globally spreading. Therefore, it becomes increasingly difficult to achieve therapeutic success by calculated antibiotic therapy. Consequently, rapid antibiotic resistance testing is essential. Various molecular and mass spectrometry-based approaches have been introduced in diagnostic microbiology to speed up the providing of reliable resistance data. PCR- and sequencing-based approaches are the most expensive but the most frequently applied modes of testing, suitable for the detection of resistance genes even from primary material. Next generation sequencing, based either on assessment of allelic single nucleotide polymorphisms or on the detection of nonubiquitous resistance mechanisms might allow for sequence-based bacterial resistance testing comparable to viral resistance testing on the long term. Fluorescence in situ hybridization (FISH), based on specific binding of fluorescence-labeled oligonucleotide probes, provides a less expensive molecular bridging technique. It is particularly useful for detection of resistance mechanisms based on mutations in ribosomal RNA. Approaches based on MALDI-TOF-MS, alone or in combination with molecular techniques, like PCR/electrospray ionization MS or minisequencing provide the fastest resistance results from pure colonies or even primary samples with a growing number of protocols. This review details the various approaches of rapid resistance testing, their pros and cons, and their potential use for the diagnostic laboratory.

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Rapid Detection of New Delhi Metallo-β-Lactamase Gene and Variants Coding for Carbapenemases with Different Activities by Use of a PCR-Based In Vitro Protein Expression Method

Cited by 7 publications

References 30 publications

Assessing mycoplasma contamination of cell cultures by qPCR using a set of universal primer pairs targeting a 1.5 kb fragment of 16S rRNA genes

Assessing mycoplasma contamination of cell cultures by qPCR using a set of universal primer pairs targeting a 1.5 kb fragment of 16S rRNA genes

NDM Metallo-β-Lactamases and Their Bacterial Producers in Health Care Settings

Emerging Rapid Resistance Testing Methods for Clinical Microbiology Laboratories and Their Potential Impact on Patient Management

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