Viral Load Quantification by Digital PCR

Real-time quantitiative PCR (qPCR) offers an extremely sensitive and specific approach for viral detection, monitoring of viral contamination, and quantification of viral load—especially when compared to other techniques such as ELISA, or immunohistochemical or cell culture methods. However, there are times when qPCR results are ambiguous or inconclusive, due to limited sensitivity or precision in analyzing samples with extremely low level viral loads.

Digital PCR is a new approach for detection and quantification of viruses present at levels below the limit of detection required by conventional methods, including qPCR. Digital PCR allows you to directly measure the amount of nucleic acid, providing a more precise method than PCR. Another unique benefit of digital PCR is that it can provide absolute quantification of viral DNA or RNA—without reference to viral standards or internal controls (Figure 1).

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Video: Introduction to Digital PCR

Absolute Quantification of Viral DNA or RNA Without Reference to Viral Standards or Internal Controls

 

Figure 1: Digital PCR provides a solution to the problems of resolving ambiguous experimental results in molecular testing for viruses (A.) and obtaining viral standards (B).

Digital PCR Enables Higher Sensitivity for Viral Detection

One of the main benefits of digital PCR is that it offers researchers absolute, rather than relative viral nucleic acid quantification. This can be especially useful for samples in which virus is detected sporadically using other methods, resulting in ambiguous results. Figure 2 shows detection of HIV and HCV in human plasma samples by digital PCR using TaqMan® OpenArray® Digital PCR Kits. This experiment illustrates detection of these RNA viruses in a sample wih a high viral load (HIV) and a sample with suspected HCV, that is not reliably detected using conventional qPCR. In the suspected HCV-infected sample, TaqMan® OpenArray Plate digital PCR, using two different TaqMan® Assays targeting HCV, successfully detected and quantified the virus, even though it was present at only 1–2 copies per µL.

Figure 2: Detection of HIV and HCV by TaqMan® OpenArray® Digital PCR in human plasma samples with high and low viral loads. The SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase and three (HIV) or two (HCV) TaqMan® Assays were used for digital PCR analysis of RNA obtained from samples with high (HIV) and low (HCV) viral loads. The experiment was run using TaqMan® OpenArray® Digital PCR Plates. The amplification heatmap shows wells with detected (red) or not-detected target amplification.

Benefits of Digital PCR for Virologists

  • Quantify viral genomes per µL sample with high precision and without using a standard curve—make your own viral standard if needed
  • Detect virus even at extremely low levels—well beyond the detection range of conventional qPCR
  • Use very small samples—digital PCR requires only very small quantities of sample
  • Compare results lab-to-lab, instrument-to-instrument, and scientist-to-scientist
  • Discriminate subtle differences in viral load—adjust the number of replicate reactions to obtain the precision you need
  • Analyze data quickly and easily—OpenArray® Digital PCR Software provides rapid, simple data analysis
  • Take advantage of rapid nucleic acid isolation protocols—digital PCR dramatically reduces the impact of polymerase inhibitors in complex samples
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