Digital Droplet PCR vs RT-qPCR for the Detection of SARS-CoV-2, Target Genes

Midsection of female doctor with swab test sample during COVID-19 crisis. Female medical professional is holding test tube in hospital. She is wearing protective suit.
Researchers sought to compare the diagnostic accuracy of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) vs digital droplet PCR for the detection of SARS-CoV-2 infection among hospitalized patients.

Among hospitalized patients, digital droplet polymerase chain reaction (ddPCR) was more accurate than reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for the diagnosis of COVID-19 infection, according to study findings published in Diagnostic Microbiology & Infectious Disease.

In this study, researchers used oropharyngeal swab samples collected from patients (n=130) at a temporary COVID-19 center in China to assess the diagnostic accuracy of ddPCR vs RT-qPCR assays for the detection of SARS-CoV-2 infection. The researchers also compared the diagnostic accuracy between both assays for the detection of specific viral genes, including the internal control (IC), open reading frame (ORF), and nucleocapsid (N) genes.

Among 130 samples tested for SARS-CoV-2 infection via RT-qPCR, 89 were positive, 32 were negative, and 9 were suggestive of infection. After repeat testing via ddPCR, 93 samples were positive, 16 were negative, and 21 were suggestive of infection, indicating increased sensitivity of ddPCR vs RT-qPCR.

Stratified by viral gene targets, all 130 samples were positive for the IC gene, and 89 were positive for the ORF gene using both ddPCR and RT-qPCR assays. Of 41 samples that were negative for the ORF using RT-qPCR, 6 (14.6%) were positive with ddPCR. For the N gene, 98 samples tested positive using both methods; however, of 32 samples that tested negative with RT-qPCR, 14 (41.75%) were positive with ddPCR. Together, these data indicated that the sensitivity of ddPCR was increased compared with RT-qPCR for the detection of both ORF and N genes.

In addition, the median cycle threshold (CT) values of the IC (CT, 146.10 vs 27.20), ORF (CT, 6.42 vs 32.52), and N (CT, 8.814 vs 30.21) genes differed between the ddPCR vs RT-qPCR assays, respectively. In regard to the IC gene, CT values were increased among samples that tested negative for SARS-CoV-2 infection, indicating a decrease in total RNA content compared with positive samples.

Overall, there was significant correlation between sampling techniques for IC (R2 =0.9752), ORF (R2 =0.8455), and N (R2 =0.8942) genes.

As each SARS-CoV-2 genome has only 1 copy of the N and ORF genes, the CT values should be similar. However, the researchers observed that the sensitivity of RT-qPCR was increased for the ORF gene (P =.00046), but ddPCR tended to have increased sensitivity to the N gene (P =.062).

This study may have been limited by the number of patients who tested negative for SARS-CoV-2 infection. As all included patients were hospitalized with symptoms, the negativity rate for both assays may indicate poor sampling procedures.

The researchers concluded the ddPCR method was more sensitive compared with RT-qPCR, and although results from both assays were significantly correlated, ddPCR may be needed for a more accurate diagnosis of COVID-19 infection.


Li J, Lin W, Du P, et al. Comparison of reverse-transcription qPCR and droplet digital PCR for the detection of SARS-CoV-2 in clinical specimens of hospitalized patients. Diagn Microbiol Infect Dis. 2022;103(2):115677. doi:10.1016/j.diagmicrobio.2022.115677