Abstract
Current methods used for diagnosis of acute infection of pathogens rely on detection of nucleic acids, antigens, or certain classes of antibodies such as IgM. Here we report a virus enzyme assay as an alternative to these methods for detection of acute viral infection. In this method, we used a luciferin derivative as the substrate for detection of the enzyme activity of influenza viral neuraminidase as a means for diagnosis of influenza. The resulting commercial test, the qFLU Dx Test, uses a different supply chain that does not compete with those for the current tests. The assay reagents were formulated as a master mix that accommodated both the neuraminidase and luciferase reactions, thereby enabling rapid and prolonged production of stable light signal in the presence of influenza virus in the sample. The assay was evaluated using depository throat swab specimens. As expected, the assay exhibited similar detection rates for all influenza types and subtypes except for A(H7N9), which exhibited lower detection rate due to lower viral titer in the specimens. When throat swab specimens were diluted with the sample buffer of the test kit and tested with the qFLU Dx Test. The sensitivity and specificity were 82.41% (95% confidence interval: 79.66–85.84%) and 95.39% (95% confidence interval: 94.32–96.46%), respectively, for these diluted specimens in comparison to a real-time polymerase chain reaction assay. The uniqueness of the qFLU Dx Test as an enzymatic assay makes it highly complementary with currently available methods.
Highlights
Current methods used for diagnosis of acute infection of pathogens rely on detection of nucleic acids, antigens, or certain classes of antibodies such as IgM
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
Average signal (RLU × 1000) 1215 3073 2131 3016 356. Diagnostic assays, those that can be used in point of care settings, play an important role in pandemic and epidemic management as vividly demonstrated in the ongoing COVID-19 pandemic
Summary
Current methods used for diagnosis of acute infection of pathogens rely on detection of nucleic acids, antigens, or certain classes of antibodies such as IgM. We report a virus enzyme assay as an alternative to these methods for detection of acute viral infection In this method, we used a luciferin derivative as the substrate for detection of the enzyme activity of influenza viral neuraminidase as a means for diagnosis of influenza. 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 provide sufficient specificity of diagnosis of infection. 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.
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