Digital PCR for direct quantification of viruses without DNA extraction

Pavšič, Jernej; Žel, Jana; Milavec, Mojca

doi:10.1007/s00216-015-9109-0

Cited by 41 publications

(45 citation statements)

References 17 publications

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“…Similarly, better tolerability of reverse transcription (RT)-dPCR to PCR inhibition was shown to detect viral hepatitis A and norovirus RNA in lettuce and bottled water [24]. The higher tolerance to PCR inhibition may also allow direct quantification of viruses from samples without prior extraction, as shown by Pavšič et al on CMV viruses, yielding a higher concentration of detectable DNA copies compared with extracted DNA [21].…”

Section: Pcr Inhibitory Substancesmentioning

confidence: 90%

“…dPCR was recently described for direct quantification of viral nucleic acids without initial DNA or RNA isolation. Hence, dPCR provides a quantitative strategy that minimizes pre-PCR processing variation and prevents an excessive loss of target sequences due to the isolation procedure [21]. Alternatively, dPCR has been frequently explored for testing low-levels of b Due to the presence of a large amount of rain (grey), the distinction between negative (red) and positive partitions (green) is not clear, and setting an inappropriate threshold may result in a large number of false positives or false negatives.…”

Section: Applications Of Digital Polymerase Chain Reaction (Dpcr) In mentioning

confidence: 99%

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The Future of Digital Polymerase Chain Reaction in Virology

et al. 2016

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Driven by its potential benefits over currently available methods, and the recent development of commercial platforms, digital polymerase chain reaction (dPCR) has received increasing attention in virology research and diagnostics as a tool for the quantification of nucleic acids. The current technologies are more precise and accurate, but may not be much more sensitive, compared with quantitative PCR (qPCR) applications. The most promising applications with the current technology are the analysis of mutated sequences, such as emerging drug-resistant mutations. Guided by the recent literature, this review focuses on three aspects that demonstrate the potential of dPCR for virology researchers and clinicians: the applications of dPCR within both virology research and clinical virology, the benefits of the technique over the currently used real-time qPCR, and the importance and availability of specific data analysis approaches for dPCR. Comments are provided on current drawbacks and often overlooked pitfalls that need further attention to allow widespread implementation of dPCR as an accurate and precise tool within the field of virology.

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Section: Pcr Inhibitory Substancesmentioning

confidence: 90%

Section: Applications Of Digital Polymerase Chain Reaction (Dpcr) In mentioning

confidence: 99%

The Future of Digital Polymerase Chain Reaction in Virology

et al. 2016

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“…Because dPCR quantifies using endpoint instead of real-time amplification, quantification is less affected by inhibitors of amplification that may be present in the sample (10,11), and it is also less affected by poor amplification efficiency (4,12,13). In fact, it may be possible to perform dPCR on samples without prior extraction of the nucleic acids (14). While qPCR and dPCR are generally equally sensitive given an equivalent input of template (15), dPCR assays have been shown to quantify some targets more precisely and are especially useful for precise quantification of low viral loads for monitoring antiviral therapy (3,4,6,13,(15)(16)(17).…”

Section: Comparison Of Dpcr To Qpcrmentioning

confidence: 99%

“…Reference standards that are used in routine qPCR assays can be initially calibrated using dPCR, which does not rely on a calibrator for quantitation (6,7,9). Digital PCR has been employed to assign values to reference materials used in CMV qPCR assays (5,14,35). The National Institute of Standards and Technology recently established a new CMV standard using dPCR (36).…”

Section: Applications Of Dpcr For Clinical Microbiologymentioning

confidence: 99%

Applications of Digital PCR for Clinical Microbiology

2017

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Digital PCR (dPCR) is an important new tool for use in the clinical microbiology laboratory. Its advantages over quantitative PCR (qPCR), including absolute quantification without a standard curve, improved precision, improved accuracy in the presence of inhibitors, and more accurate quantitation when amplification efficiency is low, make dPCR the assay of choice for several specimen testing applications. This minireview will discuss the advantages and disadvantages of dPCR compared to qPCR, its applications in clinical microbiology, and considerations for implementation of the method in a clinical laboratory.

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“…Quantitation is then determined by counting the number of positive nanoscale reactions and using Poisson statistics to produce a result that no longer depends on relation to a standard curve. Digital PCR has increasingly been thought to represent a reference standard for quantitation, particularly of DNA viruses (17)(18)(19). In addition to generating single-assay commutability determinations, we introduce a novel statistical term that directly relates the concept of commutability with clinical scales, asking whether degree of commutability is a useful concept in establishing clinical significance.…”

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confidence: 99%

Quantitative Assessment of Commutability for Clinical Viral Load Testing Using a Digital PCR-Based Reference Standard

Sun

Buelow

et al. 2016

J Clin Microbiol

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c Given recent advances in the development of quantitative standards, particularly WHO international standards, efforts to better understand the commutability of reference materials have been made. Existing approaches in evaluating commutability include prediction intervals and correspondence analysis; however, the results obtained from existing approaches may be ambiguous. We have developed a "deviation-from-ideal" (DFI) approach to evaluate commutability of standards and applied it to the assessment of Epstein-Bar virus (EBV) load testing in four quantitative PCR assays, treating digital PCR as a reference assay. We then discuss advantages and limitations of the DFI approach as well as experimental design to best evaluate the commutability of an assay in practice. Quantification of viral load is integral to clinical care, particularly among immunocompromised patients (1-4). Increases in viral load may trigger preemptive therapy to prevent or treat systemic viral disease. It may also be used to monitor therapeutic response and to determine treatment endpoints. A wide variety of assays are used for these determinations (few FDA approved), and the field continues to be hindered by a lack of agreement among the results of these tests (5-8). In turn, this lack of agreement (particularly between laboratories) prevents both the development of standardized therapeutic breakpoints and the portability of patient results among institutions. Numerous factors have been shown to contribute to the variability of results (9), one of which is a lack of standardized calibration standards. The availability of WHO international quantitative standards for cytomegalovirus (CMV) and, more recently, Epstein-Barr virus (EBV) should help the process of developing such standardization; however, numerous issues remain. These issues include the development of reliable secondary standards, which are traceable to and accurately representative of the WHO standard material. As has been shown for CMV secondary standards, such trueness cannot be assumed (10).Another issue of concern is the commutability of reference materials, which focuses on whether standards behave like patient samples and are consistent across different assays. Commutability has been defined as "the equivalence of the mathematical relationships among the results of different measurement procedures for a reference material and for representative samples of the type intended to be measured" (11). First defined and accepted in the field of clinical chemistry, more recent work in clinical molecular virology demonstrated its importance to interassay agreement, with commutable standards improving and noncommutable standards diminishing quantitative agreement (12-15). Currently, commutability is typically assessed via prediction interval approaches (14) or by multivariate techniques such as correspondence analysis (16), both based on evaluating whether a reference material belongs to the same distribution obtained from measures of human specimens using two or more assays. Predicti...

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Digital PCR for direct quantification of viruses without DNA extraction

Cited by 41 publications

References 17 publications

The Future of Digital Polymerase Chain Reaction in Virology

The Future of Digital Polymerase Chain Reaction in Virology

Applications of Digital PCR for Clinical Microbiology

Quantitative Assessment of Commutability for Clinical Viral Load Testing Using a Digital PCR-Based Reference Standard

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