Detection of Methicillin-Resistant Staphylococcus aureus by a Duplex Droplet Digital PCR Assay

Kelley, KaShonda; Cosman, Angela M.; Belgrader, Phillip; Chapman, Brenda; Sullivan, Donna C.

doi:10.1128/jcm.00196-13

Cited by 67 publications

(41 citation statements)

References 43 publications

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“…dPCR has been shown to be a highly precise measurement technique, especially for relatively concentrated materials (44), and has demonstrated good accuracy for certification of standards for genetically modified organism testing (45). dPCR showed good discrimination between methicillin-resistant and methicillin-sensitive Staphylococcus aureus isolates; thus, it might enable better discrimination of different genotypes of M. tuberculosis (46). Furthermore, it has been successfully applied in a one-step format for the quantification of RNA viruses showing increased tolerance to inhibitors and higher accuracy at lower concentrations than qPCR (47).…”

Section: Development Of Reference Measurement Systems For Pathogen Naatsmentioning

confidence: 99%

Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses

Pavšič

Devonshire²,

Parkes³

et al. 2015

J Clin Microbiol

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f Nucleic acid-based tests for infectious diseases currently used in the clinical laboratory and in point-of-care devices are diverse. Measurement challenges associated with standardization of quantitative viral load testing are discussed in relation to human cytomegalovirus, BK virus, and Epstein-Barr virus, while the importance of defining the performance of qualitative methods is illustrated with Mycobacterium tuberculosis and influenza virus. The development of certified reference materials whose values are traceable to higher-order standards and reference measurement procedures, using, for instance, digital PCR, will further contribute to the understanding of analytical performance characteristics and promote clinical data comparability. Molecular approaches, such as nucleic acid (NA) amplification-based tests (NAATs) and sequence analysis, are increasingly replacing conventional microbiological methods, such as pathogen propagation in culture and techniques for the detection of antigens, for (i) viral load quantification, (ii) the detection of pathogenic viruses and bacteria, (iii) monitoring viral and bacterial resistance to therapeutic agents, and (iv) monitoring transmission across communities, as they often enable faster, more accurate, or more sensitive measurements (1, 2). However, commercial and in-house NAATs for infectious diseases utilize different technologies, reaction chemistries, and calibration materials, leading to demonstrable variability in the test results in terms of (i) numerical values (e.g., numbers of genome copies) for quantitative methods or (ii) presence or absence of the pathogen for qualitative methods (3-5).The In Vitro Diagnostics Directive (IVDD) in Europe has promoted assay standardization in the clinical laboratory community, through requiring manufacturers to provide information about the traceability of their calibrators (for definitions of terms in italics, see Table 1), in compliance with ISO 17511 (6). The concept of metrological traceability in clinical chemistry and laboratory medicine has been comprehensively discussed in several articles (7-9). For small-molecule measurements in clinical chemistry, such as those of blood glucose, creatinine, cortisol, and electrolytes, reference measurement procedures of high metrological quality are available which relate the quantity value of the calibrators and reference materials used in a calibration hierarchy traceable to the International System of Units (SI) as described in ISO 17511 (6, 8). However, for the majority of the more complex biomeasurements, including those based on NA testing and infectious pathogen detection, reference materials whose values are traceable to the SI and reference measurement procedures (with well-understood uncertainty) are not yet available. ISO 17511 indicates recognition of the fact that, for measurements of, for example, viral NAs (for human cytomegalovirus [CMV], human immunodeficiency virus [HIV], and hepatitis B virus [HBV]), typically WHO international standards (IS) are available, w...

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Section: Development Of Reference Measurement Systems For Pathogen Naatsmentioning

confidence: 99%

Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses

Pavšič

Devonshire²,

Parkes³

et al. 2015

J Clin Microbiol

View full text Add to dashboard Cite

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“…This includes the quantification of free tumor DNA (tDNA) or microRNAs (miRNAs) from plasma specimens as an indicator of some types of carcinomas and a method to simultaneously screen for and quantify 4 common mutations in the KRAS gene in serum specimens (127,(141)(142)(143). Digital PCR has also been used in infectious disease for detection of S. aureus and C. trachomatis (144,145) and has demonstrated the ability to accurately quantify HIV and hepatitis C virus (HCV) loads over a wide dynamic range (146,147). A very recent publication has demonstrated the utility of dPCR to differentiate between HHV-6 reactivation and chromosomally integrated HHV-6 (ciHHV-6) in pre-and posttransplant patients (148).…”

Section: Digital Pcrmentioning

confidence: 99%

Emerging Technologies for the Clinical Microbiology Laboratory

2014

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SUMMARY In this review we examine the literature related to emerging technologies that will help to reshape the clinical microbiology laboratory. These topics include nucleic acid amplification tests such as isothermal and point-of-care molecular diagnostics, multiplexed panels for syndromic diagnosis, digital PCR, next-generation sequencing, and automation of molecular tests. We also review matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry methods and their role in identification of microorganisms. Lastly, we review the shift to liquid-based microbiology and the integration of partial and full laboratory automation that are beginning to impact the clinical microbiology laboratory.

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“…16 Alternatives to these options have emerged in recent years, such as the use of electrochemical sensors. They were found to be superior to the conventional approaches, but require complicated electrode modification.…”

Section: Introductionmentioning

confidence: 99%

A Graphene Oxide–Based Sensing Platform for the Determination of Methicillin-Resistant Staphylococcus aureus Based on Strand-Displacement Polymerization Recycling and Synchronous Fluorescent Signal Amplification

et al. 2016

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To develop new technology for detecting methicillin-resistant Staphylococcus aureus (MRSA), a novel fluorescent biosensor based on Klenow fragment (KF)-assisted target recycling amplification and synchronous fluorescence analysis was created. Carboxy-fluorescein (FAM)-labeled single-stranded DNA (ssDNA) containing a capture probe and a signal probe was adsorbed onto the surface of graphene oxide (GO) via π-stacking interactions, resulting in the fluorescence quenching of the dye. When target and primer were introduced, the fluorescence was restored due to P0 being completely released from the surface of the GO. Meanwhile, by using the KF and exploiting the synergistic effect of FAM and the doublestranded DNA (dsDNA)-SYBR Green I duplex structure, the fluorescence in this detection system was considerably amplified and the sensitivity was improved. The proposed strategy for mecA gene analysis showed a good linear range from 1 to 40 nmol/L, with a lower limit of detection of 0.5 nmol/L. In addition, a bacterial sample harboring the mecA gene was also detected, and its lower detection limit was up to 300 colony-forming units (CFU)/mL. Accordingly, this biosensor exhibits high sensitivity and selectivity and has great potential for early clinical diagnosis and treatment.

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Detection of Methicillin-Resistant Staphylococcus aureus by a Duplex Droplet Digital PCR Assay

Cited by 67 publications

References 43 publications

Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses

Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses

Emerging Technologies for the Clinical Microbiology Laboratory

A Graphene Oxide–Based Sensing Platform for the Determination of Methicillin-Resistant Staphylococcus aureus Based on Strand-Displacement Polymerization Recycling and Synchronous Fluorescent Signal Amplification

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