The ongoing COVID-19 pandemic reveals several significant challenges to diagnostic assays. Current methods rely on detection of viral nucleic acids, antigens, or specific antibodies, which tend to compete with the same sources of key materials and lead to shortage of supplies. Moreover, the SARS-CoV-2 virus mutates rapidly, which may affect the efficacy of these diagnostic assays. Here we propose an alternative method for detection of acute infection of a pathogen. Many viral pathogens contain unique enzymes, the activities of which may be effective diagnostic markers. Some of these enzymes had been successfully used as therapeutic drug targets, indicating that they should also provide sufficient specificity of diagnosis of infection.
Here we report a biochemiluminescent assay for detection of influenza viral neuraminidase (NA) activity as a means for detection of influenza. Influenza viral neuraminidase is an effective target of a newer generation of influenza therapeutic drugs that inhibit the activity of this enzyme, suggesting that a unique and specific substrate can be designed for use in diagnosis. A substrate derivatized with firefly luciferin improves the sensitivity considerably. Further improvement makes the assay suitable for use in point of care settings.
Like SARS-CoV-2 virus, influenza virus constantly mutates itself, and can easily be transmitted from one individual to another, annually affecting 5–10% adults and 20–30% children in the world1,2. It is estimated that globally, annual epidemics of influenza lead to 3–5 million cases of severe illness, which may require hospitalization, and about 250,000–500,000 deaths3. An uncontained pandemic influenza may lead to far worse consequences4. Rapid diagnosis of epidemic or pandemic influenza plays an important role in effective management of influenza and public health.
Rapid influenza diagnosis tests (RIDTs) intended for detection of active infection, which can be completed within 30 min, fall into two categories, i.e., those that detect viral antigens and those that detect viral nucleic acids. The antigen assays have a number of drawbacks, the most significant one being lack of sensitivity4,5,6,7,8. Molecular-based influenza assays, which detect the viral nucleic acids, offer better sensitivity. Among these tests, however, only a few can be completed within 30 min9,10,11,12,13,14 and thus suitable for point of care use. Antigen and nucleic acid assays suffer from a common drawback in that they all rely on static epitopic or genetic sequences, which are susceptible to mutations. Indeed, the lateral flow based RIDTs were found to be less sensitive for detection of the pH1N1 virus during the H1N1 pandemic15.
Influenza viral NA activity as a diagnostic marker for influenza had been previously explored. A colorimetric substrate specific for influenza viral NA was developed and successfully used in an assay for detection of influenza, demonstrating the feasibility of this strategy16,17,18,19,20. A more sensitive chemiluminescent substrate derivatized with dioxetane was subsequently developed and used in a test for influenza diagnosis21,22. Although the chemiluminescent assay had 88% clinical sensitivity in comparison to culture method when nasal aspirate specimens were used23,24, the assay was too complex for it to be widely adopted for clinical use, particularly for point of care use.
Here we report a biochemiluminescent assay using a luciferin derivatized substrate for specific detection of influenza viral NA activity3,25,26. The assay reagents were formulated as a lyophilized master mix to simplify the assay procedure, which essentially is a one-step assay (Fig. 1). Presence of influenza viral NA in the reaction generates stable light signal, which lasts for at least 2 h. The stable light signal can be detected using a simple luminometer. The entire detection procedure takes approximately 15 min. Because firefly luciferase/luciferin is a highly sensitive biochemiluminescence, the qFLU Dx Test is sensitive. The present work demonstrated the feasibility of using viral enzyme activity as a means for detection of acute viral infection.
Biochemiluminescence Reaction. The qFLU Dx Test uses 4, 7-dimethyl neuraminic acid -O-luciferin as the substrate for detection of influenza virus. In the presence of Type A or B influenza virus, viral neuraminidase cleaves the substrate to release the luciferin moiety, which is oxidized to oxyluciferin by luciferase to generate light signal. Since the substrate is continuously cleaved the neuraminidase to result in luciferin that is continuously oxidized to generate light signal, this is a “real time” assay with glow light feature.
