Review
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 01/13/15 For personal use only.
The potential advantages of digital PCR for clinical virology diagnostics Expert Rev. Mol. Diagn. 14(4), 501–507 (2014)
Ruth Hall Sedlak1 and Keith R Jerome*1,2 1 Department of Laboratory Medicine, University of Washington, Seattle, Washington 98195, USA 2 Program in Infectious Diseases, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, 98109, USA *Author for correspondence: Tel.: +1 206 667 6793 Fax: +1 206 667 4411 [email protected]
Digital PCR (dPCR), a new nucleic acid amplification technology, offers several potential advantages over real-time or quantitative PCR (qPCR), the current workhorse of clinical molecular virology diagnostics. Several studies have demonstrated dPCR assays for human cytomegalovirus or HIV, which give more precise and reproducible results than qPCR assays without sacrificing sensitivity. Here we review the literature comparing dPCR and qPCR performance in viral molecular diagnostic assays and offer perspective on the future of dPCR in clinical virology diagnostics. KEYWORDS: accuracy • digital PCR • precision • sensitivity • viral quantitation
PCR technologies in viral diagnostics
Nucleic acid amplification techniques (NAATs) form the cornerstone of clinical viral diagnostics. PCR and quantitative or real-time PCR (qPCR) have been widely utilized to diagnose viral disease from plasma/serum, blood, cerebrospinal fluid and tissue samples. During qPCR, target template DNA is amplified with sequence-specific primers until it produces a signal that is supplied through a DNA intercalating dye or sequence-specific fluorescent probe. Higher levels of template DNA produce a fluorescent signal that is detectable after fewer cycles of PCR, and a standard curve is used to correlate the cycle giving a signal with a known quantity of DNA [1]. This technique has served the field well but has some drawbacks; namely, the necessity for a standard curve and the lack of universal standards of known quantity, both of which contribute to intra- and interlaboratory imprecision and lack of commutability. Recently, a NAAT called digital PCR (dPCR) has become commercially available, and its utility in viral diagnostics has been explored by several clinical virology laboratories. dPCR uses the same primers and dyes/ probes as qPCR, but partitions a single bulk reaction into thousands or millions of separate microliter to picoliter scale reactions [2,3]. Each of these reactions is cycled to the endpoint of informahealthcare.com
10.1586/14737159.2014.910456
the reaction, and each partition is read as positive or negative for target template DNA. Using Poisson statistics, the number of positive partitions is compared with the total number of partitions to give a direct readout or absolute quantitation of the number of target DNA template molecules in the reaction, abrogating the need for the standard curve traditionally used in qPCR [4,5]. Several commercial platforms with reasonable throughput are available for dPCR applications, making this a practical option for certain applications in clinical laboratories. Platforms from Fluidigm Corporation and Life Technologies utilize a plate format to partition reactions in separate wells, while platforms available from Bio-Rad Laboratories and RainDance use droplets formed by a water-inoil emulsion to partition reactions (known as droplet dPCR, ddPCR). The per sample cost of Bio-Rad’s ddPCR system is currently the lowest of all the systems at $3–5 per well. Details of these systems comparing number of partitions and estimated costs are reviewed elsewhere [6,7]. While qPCR is the current gold standard for molecular quantitation in viral diagnostics, dPCR offers several potential advantages over qPCR. Here we will review the literature addressing how dPCR could improve upon current viral diagnostic quantitation capabilities by increasing precision, reproducibility
2014 Informa UK Ltd
ISSN 1473-7159
501
Review
Sedlak & Jerome
Table 1. Imprecision of ddPCR and quantitative PCR measured at several dilutions of cytomegalovirus-positive patient plasma sample.
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 01/13/15 For personal use only.
Coefficient of variation (%) Copies/ml
ddPCR
qPCR
N
16,500
3.4
12.1
8
3830
8.0
17.8
8
1604
11.8
20.0
8
480
10.0
13.4
8
ddPCR: Droplet digital PCR; qPCR: Quantitative PCR.
and sensitivity, and by standardizing quantitation within the field. We will also discuss limitations of the current generation of dPCR platforms that may limit their widespread immediate adoption into the clinical laboratory. dPCR exhibits precision superior to qPCR
The imprecision of typical clinical viral qPCR assays can be quite high, particularly at lower template copy numbers. Even within highly specialized laboratories, the coefficient of variation (CV, a measure of imprecision) can be 20–30% or higher [8–12]. The clinical impact of this variation in measurement is not fully known, but several studies on cytomegalovirus (CMV) and HIV have suggested that increases in viral load even at very low levels can be clinically significant [13–16]. For example, a study by Waggoner et al. comparing the performance of a commercially available CMV qPCR assay with the laboratory’s reference protocol explored the clinical significance of low CMV viral loads (
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 01/13/15 For personal use only.
The potential advantages of digital PCR for clinical virology diagnostics Expert Rev. Mol. Diagn. 14(4), 501–507 (2014)
Ruth Hall Sedlak1 and Keith R Jerome*1,2 1 Department of Laboratory Medicine, University of Washington, Seattle, Washington 98195, USA 2 Program in Infectious Diseases, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, 98109, USA *Author for correspondence: Tel.: +1 206 667 6793 Fax: +1 206 667 4411 [email protected]
Digital PCR (dPCR), a new nucleic acid amplification technology, offers several potential advantages over real-time or quantitative PCR (qPCR), the current workhorse of clinical molecular virology diagnostics. Several studies have demonstrated dPCR assays for human cytomegalovirus or HIV, which give more precise and reproducible results than qPCR assays without sacrificing sensitivity. Here we review the literature comparing dPCR and qPCR performance in viral molecular diagnostic assays and offer perspective on the future of dPCR in clinical virology diagnostics. KEYWORDS: accuracy • digital PCR • precision • sensitivity • viral quantitation
PCR technologies in viral diagnostics
Nucleic acid amplification techniques (NAATs) form the cornerstone of clinical viral diagnostics. PCR and quantitative or real-time PCR (qPCR) have been widely utilized to diagnose viral disease from plasma/serum, blood, cerebrospinal fluid and tissue samples. During qPCR, target template DNA is amplified with sequence-specific primers until it produces a signal that is supplied through a DNA intercalating dye or sequence-specific fluorescent probe. Higher levels of template DNA produce a fluorescent signal that is detectable after fewer cycles of PCR, and a standard curve is used to correlate the cycle giving a signal with a known quantity of DNA [1]. This technique has served the field well but has some drawbacks; namely, the necessity for a standard curve and the lack of universal standards of known quantity, both of which contribute to intra- and interlaboratory imprecision and lack of commutability. Recently, a NAAT called digital PCR (dPCR) has become commercially available, and its utility in viral diagnostics has been explored by several clinical virology laboratories. dPCR uses the same primers and dyes/ probes as qPCR, but partitions a single bulk reaction into thousands or millions of separate microliter to picoliter scale reactions [2,3]. Each of these reactions is cycled to the endpoint of informahealthcare.com
10.1586/14737159.2014.910456
the reaction, and each partition is read as positive or negative for target template DNA. Using Poisson statistics, the number of positive partitions is compared with the total number of partitions to give a direct readout or absolute quantitation of the number of target DNA template molecules in the reaction, abrogating the need for the standard curve traditionally used in qPCR [4,5]. Several commercial platforms with reasonable throughput are available for dPCR applications, making this a practical option for certain applications in clinical laboratories. Platforms from Fluidigm Corporation and Life Technologies utilize a plate format to partition reactions in separate wells, while platforms available from Bio-Rad Laboratories and RainDance use droplets formed by a water-inoil emulsion to partition reactions (known as droplet dPCR, ddPCR). The per sample cost of Bio-Rad’s ddPCR system is currently the lowest of all the systems at $3–5 per well. Details of these systems comparing number of partitions and estimated costs are reviewed elsewhere [6,7]. While qPCR is the current gold standard for molecular quantitation in viral diagnostics, dPCR offers several potential advantages over qPCR. Here we will review the literature addressing how dPCR could improve upon current viral diagnostic quantitation capabilities by increasing precision, reproducibility
2014 Informa UK Ltd
ISSN 1473-7159
501
Review
Sedlak & Jerome
Table 1. Imprecision of ddPCR and quantitative PCR measured at several dilutions of cytomegalovirus-positive patient plasma sample.
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 01/13/15 For personal use only.
Coefficient of variation (%) Copies/ml
ddPCR
qPCR
N
16,500
3.4
12.1
8
3830
8.0
17.8
8
1604
11.8
20.0
8
480
10.0
13.4
8
ddPCR: Droplet digital PCR; qPCR: Quantitative PCR.
and sensitivity, and by standardizing quantitation within the field. We will also discuss limitations of the current generation of dPCR platforms that may limit their widespread immediate adoption into the clinical laboratory. dPCR exhibits precision superior to qPCR
The imprecision of typical clinical viral qPCR assays can be quite high, particularly at lower template copy numbers. Even within highly specialized laboratories, the coefficient of variation (CV, a measure of imprecision) can be 20–30% or higher [8–12]. The clinical impact of this variation in measurement is not fully known, but several studies on cytomegalovirus (CMV) and HIV have suggested that increases in viral load even at very low levels can be clinically significant [13–16]. For example, a study by Waggoner et al. comparing the performance of a commercially available CMV qPCR assay with the laboratory’s reference protocol explored the clinical significance of low CMV viral loads (
