Digital PCR is a method for absolute nucleic acid quantification. Individual analyte molecules are partitioned into many replicate reactions diluted to the point where one or zero molecules are present in most reactions. After endpoint PCR, the starting template concentrations is calculated using Poisson statistical analysis of the number of reactions that contained amplified target and were positive and the number that were negative where no target was detected. Digital PCR is often compared to real-time PCR also known as quantitative PCR (qPCR). In qPCR DNA is replicated until it reaches a certain level of signal and the number of amplification cycles needed to reach this point is used to calculate how many template DNA molecules were originally present relative to other DNA molecules in the sample.
Applications of dPCR include distinguishing of differential expression of alleles inherited from each parent, quantify cancer genes in patient specimens and detection of fetal DNA in circulating blood. The technique digital PCR was first described in 1992 by researchers in Australia and named by Bert Vogelstein and Ken Kinzler at Johns Hopkins University and demonstrated use of the technique to quantify disease-associated mutations in stool samples from patients with colorectal cancer. Digital PCR was first commercialized in 2006 by Fluidigm. Nanofabrication and microfluidics approaches allow digital PCR systems to have hundreds to millions of nanoliter- or picoliter-scale partitions using chips or plates.
The combination of nanoliter-sized droplets with digital PCR is called droplet digital PCR (ddPCR). In ddPCR, samples are mixed with reagents and dispersed into tiny aqueous droplets within oil that are transferred into tubes that are placed in a thermocycler for PCR. The tubes are then transferred to a droplet reading machine that works like a flow cytometer to analyze whether or not a reaction occurred in each droplet.
Digital PCR and ddPCR is being applied to the development of diagnostic and monitoring of coronavirus SARS-CoV-2 which causes COVID-19 disease. A research group at Nantong University, China reported using digital PCR to detect coronavirus SARS-CoV-2 in patient samples with less false negative and false positive results compared with real time PCR (RT-PCR), especially for samples with low viral load. Researchers at Capital Medical University, Beijing, used ddPCR for quantitative monitoring of viral load in COVID-19 patients. Researchers conducted at the National Institute of Metrology, Beijing, found digital PCR to be accurate and sensitive for detection of SARS-CoV-2. Researchers based at Wuhan University reported ddPCR to be more accurate and sensitive than RT-PCR in samples with low viral load.
Gnomegen, a San Diego-based company, received an emergency use authorization from the FDA for its COVID-19 RT-Digital PCR Detection kit in April, 2020. Israel-based company, Todos Medical, acquired the exclusive distribution rights for Gnomegen’s digital PCR and qPCR SARS-CoV-2 tests in North America, Europe, Southeast Asia and Latin America.
Digital PCR technology has been applied to monitoring the environment for SARS-CoV-2 virus. During the COVID-19 pandemic ddPCR was used to test for SARS-CoV-2 viral RNA in aeorsols collected on gelatin filters in patient and medical staff areas at Renmin Hospital of Wuhan University, Wuchang Fangcang Field Hospital and public areas in Wuhan.
