Digital droplet PCR is a promising novel technology for the detection and quantitation of viral genetic material. In this study, we demonstrate that the platform is generally comparable to traditional RT-PCR methods for both total HIV-DNA and episomal 2-LTR circles. Despite good correlations of both linear and episomal DNA standards between the two methods, differences in the number of genomic HIV-1 DNA copies and episomal 2-LTR circles were observed. Whereas ddPCR is a direct measure of target DNA concentration, real-time PCR relies on a standard curve to extrapolate the amount of target present. The PCR quantitation threshold cycle (Ct) of an unknown sample is compared to the Ct of known input/expected copy number of a dilutional standard. The copy number of the standard is calculated prior to PCR quantitation by measuring concentrations of a known linear or episomal DNA sequence with a known molecular mass. One potential cause of the discrepancy between the assays could be errors introduced by spectrophotometric determination of the DNA concentration, or subsequent serial dilution of the standards. It is also possible that PCR was less efficient under one set of PCR conditions, or that some of the genomic DNA remained undigested prior to droplet generation. Larger fragments may not have packaged efficiently into picoliter droplets, leading to an underestimation of HIV-1 DNA copies by ddPCR. By contrast, real-time PCR quantitation most likely would not be affected by size differences in genomic DNA strands as all genetic material is included in each reaction without packaging or partitioning.
Discrepancies between the two PCR approaches in measuring the number of 2-LTR circles may have been related to the fact that the input copy numbers were low overall. Despite relatively good linearity at low standard copy numbers for both real-time and ddPCR, a relatively small number of positive ddPCR droplets could dramatically alter the results in low copy-number samples. For example, 63 LTR circles/million PBMCs were quantified by ddPCR for one patient, but only 2.3 copies/million PBMCs by real-time PCR. There was, however, a high degree of variation between the observed copy numbers of the duplicate samples evaluated by ddPCR for this patient (3.7 and 123.6 copies). Furthermore, the maximum volume of input sample in ddPCR is 7.5 μL, whereas a larger sample volume can be included in real-time PCR. As a result, higher concentrations of DNA substrate may be required in ddPCR reaction wells in order to maximize sensitivity for detecting HIV-1 DNA at very low levels.
One inherent limitation of ddPCR as compared to RT-PCR is the need to dilute samples with more than 75,000 copies of the target DNA, as overloading the picoliter droplets results in a significant loss of linearity at higher copy numbers. While this problem has less impact on quantitation at the low copy numbers that would be expected for HIV-1 DNA (Richman et al., 2009
; Siliciano et al., 2003
), human chromosomal and mitochondrial DNA require significant dilution prior to assay.
Whereas ddPCR is a promising platform for the detection of total DNA, episomal DNA and viral RNA, the technology may not be compatible with certain methods used to detect integrated HIV-1 DNA without involving standard curves. For example, Alu
-PCR has become a standard technique to quantitate the integrated, proviral HIV-1 DNA fraction (Brussel, Delelis, and Sonigo, 2005
; Liszewski, Yu, and O'Doherty, 2009
-PCR relies on two PCR steps: the first amplifies the sequences between the nearest chromosomal Alu
element and integrated HIV-1 DNA. However, because the target sequence is randomly inserted between Alu
elements, different length strands from first-round PCR are created. The second step involves amplification and real-time PCR quantitation of only the HIV-1 sequence. However, this method requires a PCR standard generated by infection of a cell line containing randomly integrated HIV-1 DNA as input into the first-round PCR (Brussel, Delelis, and Sonigo, 2005
; Liszewski, Yu, and O'Doherty, 2009
). Although ddPCR could be used to quantitate HIV-1 DNA from the second PCR step, the need persists for an input standard.
Despite these limitations, ddPCR is a promising tool for the study of HIV-1 reservoirs and persistence. ddPCR and real-time PCR have similar dynamic ranges for linear and episomal DNA standards and ddPCR is generally comparable to real-time PCR for the detection and quantitation of HIV-1-DNA and 2-LTR circles. However, PCR inhibition during quantitation of large amounts of DNA may be reduced by the partitioning reactions into droplets, allowing for investigation of very low levels of HIV-1 without the use of repetitive sampling required by standard quantitative PCR methods. Employing statistical methods to define positive-negative cutoffs rather than relying on manual methods may also enhance ddPCR, and further optimization of this novel technology would improve the detection of very low-level viral genetic targets.