Researchers at Colorado State University have developed a novel nuclease protection ELISA (NP-ELISA) that has clinical relevance as an alternative to real time RT-PCR. The assay has excellent specificity with highly similar sequences and is compatible with multiple signal visualization modalities.
Many viral diseases including Zika virus, influenza, dengue virus, and chikungunya virus present with general, nonspecific symptoms that encumber differential diagnosis. Thus, the Center for Disease Control (CDC) typically recommends NAT on serum, urine, or other biologically-relevant samples to diagnose viral disease during the early state of infection – typically done with approved real-time polymerase chain reaction (PCR) assays.
Early and accurate diagnosis is crucial to monitor infection outcomes and provide timely interventions. However, gold standard PCR assays are labor-intensive and require expensive reagents and instrumentation. Nuclease protection has been used for decades to detect and quantify nucleic acid but has not yet been investigated as a diagnostic tool for infectious disease.
Researchers at Colorado State University have developed a nuclease protection-ELISA (NP-ELISA) for the specific and sensitive detection of nucleic acid. In contrast to the nuclease protection sandwich hybridization assay (NPA-SH), the NP-ELISA uses a single oligo capture probe.
Figure 1. (below) Demonstrates a probe designed to have specificity towards a respective Zika (ZIKV) or Kunjin (KUNV) virus sequence. The capture probe is mixed with a nucleic acid target (i) and hybridized products (ii) are immobilized to the bottom of a microtiter plate and are subjected to a digestion reaction with S1 nuclease which degrades single stranded nucleic acid including unbound probe (iii). HRP-conjugated anti-Digoxigenin antibody binds to a digoxigenin molecule bound to the 3’ end of the capture probe and facilitates an enzymatic readout (iv).
The assay was validated using synthesized target oligos and then compared for colorimetric, chemiluminescent, and electrochemical detection methods. The NP-ELISA is a new valuable approach for NAT that uses fewer reagents and inexpensive instrumentation compared to real-time PCR.
Collaboration and Funding Interest Welcome
Brian Geiss
Chuck Henry
Jessica Filer