Detecting Intact Virus Using Exogenous Oligonucleotide Labels

The COVID-19 pandemic has revealed how an emerging pathogen can cause a sudden and dramatic increase in demand for viral testing. Testing pooled samples could meet this demand; however, the sensitivity of reverse transcription quantitative polymerase chain reaction (RT-qPCR), the gold standard, significantly decreases with an increasing number of samples pooled. Here, we introduce detection of intact virus by exogenous-nucleotide reaction (DIVER), a method that quantifies intact virus and is robust to sample dilution. As demonstrated using two models of severe acute respiratory syndrome coronavirus 2, DIVER first tags membraned particles with exogenous oligonucleotides, then captures the tagged particles on beads functionalized with a virus-specific capture agent (in this instance, angiotensin-converting enzyme 2), and finally quantifies the oligonucleotide tags using qPCR Using spike-presenting liposomes and spike-pseudotyped lentivirus, we show that DIVER can detect 1 x 10(5) liposomes and 100 plaque-forming units of lentivirus and can successfully identify positive samples in pooling experiments. Overall, DIVER is well positioned for efficient sample pooling and clinical validation.

Automated summary

The COVID-19 pandemic has highlighted the need for efficient viral testing. The DIVER method introduced in this study can accurately quantify intact viruses and is robust to sample dilution, making it suitable for sample pooling and clinical validation.