One-step RT-droplet digital PCR: a breakthrough in the quantification of waterborne RNA viruses

Rački, Nejc; Morisset, Dany; Gutiérrez-Aguirre, Ion; Ravnikar, Maja

doi:10.1007/s00216-013-7476-y

Cited by 122 publications

(88 citation statements)

References 24 publications

(37 reference statements)

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“…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). Some limitations in the clinical sensitivity of dPCR have been observed which may be linked to restricted sample volume inputs for dPCR platforms (44).…”

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

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“…The parasite is a significant threat to water utilities 69 as it has a low infectious dose (10-100 oocysts), is able to survive 70 for long periods in the environment and is resistant to drinking 71 water disinfectants (Fayer, 2004 where the signal increases above background (Hindson et al, 87 2011;Baker, 2012). qPCR enables detection and quantitation of 88 the target nucleotide sequences, initially present in the reaction 89 mixture, down to one or a few copies (Rački et al, 2014).…”

mentioning

confidence: 99%

Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples

Yang

Paparini

Monis

et al. 2014

International Journal for Parasitology

163

127

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“…The method was transferred from a previously published qPCR assay [ 22 ]. There are also examples in the literature of one step RT-ddPCR assays that target RNA pathogens, such as Rotavirus [ 10 ] and Pepper Mild Mottle virus ( [ 12 ] and Fig. 4 ).…”

Section: Pcr Amplifi Cationmentioning

confidence: 99%

“…The variation of signals depends on many factors, e.g., assay characteristics [ 18 ], matrix or presence of inhibitors [ 10 ], single nucleotide polymorphisms (SNP) in the probe annealing region (Fig. 5 ) and others.…”

Section: Notesmentioning

confidence: 99%

“…We found that optimized qPCR assays could be easily transferred to ddPCR format. In these assays, ddPCR offered higher quantifi cation accuracy at lower concentrations [ 10 , 17 , 18 ] and lower sensitivity to inhibitors [ 10 ]. It has also been demonstrated that RNA quantifi cation is possible using a one-step reverse transcriptionddPCR (RT-ddPCR) [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

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Droplet Digital PCR for Absolute Quantification of Pathogens

Gutiérrez-Aguirre

Rački

Dreo

et al. 2015

Methods in Molecular Biology

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The recent advent of different digital PCR (dPCR) platforms is enabling the expansion of this technology for research and diagnostic applications worldwide. The main principle of dPCR, as in other PCR-based methods including quantitative PCR (qPCR), is the specific amplification of a nucleic acid target. The distinctive feature of dPCR is the separation of the reaction mixture into thousands to millions of partitions which is followed by a real time or end point detection of the amplification. The distribution of target sequences into partitions is described by the Poisson distribution, thus allowing accurate and absolute quantification of the target from the ratio of positive against all partitions at the end of the reaction. This omits the need to use reference materials with known target concentrations and increases the accuracy of quantification at low target concentrations compared to qPCR. dPCR has also shown higher resilience to inhibitors in a number of different types of samples. In this chapter we describe the droplet digital PCR (ddPCR) workflow for the detection and quantification of pathogens using the droplet digital Bio-Rad platform QX100. We present as an example the quantification of the quarantine plant pathogenic bacterium, Erwinia amylovora.

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One-step RT-droplet digital PCR: a breakthrough in the quantification of waterborne RNA viruses

Cited by 122 publications

References 24 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

Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples

Droplet Digital PCR for Absolute Quantification of Pathogens

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