Are real-time and droplet-digital polymerase chain reaction qualitative results comparable in detecting Bovine Viral Diarrhea Virus ribonucleic acid?

Using Polymerase Chain Reaction (PCR) or Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) to detect viral agents during the acute phase of infection is becoming increasingly common. To ensure the accuracy of these in vitro diagnostic tests, studies are ongoing to develop Reference Materials (RM) for molecular assays, using Bovine Viral Diarrhea Virus (BVDV) as a model. Qualitative methods for detecting BVDV ribonucleic acid (RNA) are employed to diagnose the disease in animals and to detect contamination in cell cultures. To establish a reliable routine for detection of BVDV RNA, a commercial Reverse Transcription quantitative PCR (RT-qPCR) was briefly compared to Reverse Transcriptase Droplet Digital PCR (RT-ddPCR, a primary method) using the same primers and probe. Using both methods, serial dilutions of BVDV RNA standard and BVDV RNA grown in cell culture were evaluated, with RT-qPCR showing 100 % sensitivity, with specificities of 83 % and 67 %, respectively. Discrepancies between the two techniques were observed at low RNA concentrations, with possible false positives in RT-qPCR. This finding highlights the importance of stringent validation in molecular in vitro diagnostic (IVD) methods.