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Turnkey Detection: Rapid Diagnostics in the Field

Viruses can live on surfaces, such as counters and doorknobs, for more than two hours making them highly contagious when in confined quarters. As warfighters are frequently in these types of environments, identifying viral threats rapidly is vital to treatment and halting transmission. However, a versatile, user-friendly detection and diagnostic device for multiple pathogens does not currently exist.

In an effort funded by the Defense Threat Reduction Agency’s Joint Science and Technology Office, Dr. Steven Benner from the Foundation for Applied Molecular Evolution (FfAME), is working to change this by developing a turnkey, adaptable and cost-effective device to test for several pathogens in a single assay. The device will not require laboratory diagnostic tools and can be used with minimal training.

This research represents a significant improvement in available diagnostics and surveillance kits that rely on polymerase chain reactions which require skilled interpretive analysis, specialized equipment and laboratory testing environments. DTRA’s new capability will address the existing need for increased warfighter protection from known and emerging viral pathogens at the point of infection.

Diagnosis cannot rely solely on symptoms since indicators of emerging diseases are difficult to identify. Further, a patient’s antibody response can present weeks after an active infection and persist long after the infection is resolved. Also, diagnostic tools that require laboratory analysis waste valuable time, making quarantines ineffective.

Instead, health care providers need an assay that detects the nucleic acids, the DNA or RNA from the pathogen, in both biological and environmental samples at the point of care, offering a flexible “single source” solution. A tool that can detect multiple diseases would be valuable not only in terms of warfighter protection, but in terms of affordability. Costing upwards of $200 a test, traditional diagnostic tools are expensive.

In addition, the nucleic acid-targeted diagnostic must be adaptable. As new pathogens emerge, the diagnostic capability needs to be incorporated into the existing kit. This has been a challenge to scientists working diligently to create a solution.

Recently reported in the Journal of Virological Methods article “Standard and AEGIS Nicking Molecular Beacons Detect Amplicons from the Middle East Respiratory Syndrome Coronavirus,” the on-site reporting capability showed the adaptability of the diagnostic tool to detect a coronavirus (CoV) that causes the Middle East Respiratory Syndrome (MERS).

Dr. Benner utilized a previously discovered DTRA-supported tool, the artificially expanded genetic information system (AEGIS), to create the new, high sensitivity diagnostic tool with low noise. AEGIS works by adding nucleotides to the four found in standard DNA and RNA, pairing orthogonally to the A:T and G:C pairs.

Molecular beacons containing AEGIS then generate florescent light signals, detectable at points of sampling, where it can be read on the spot or captured by a cell phone camera for transmission to a remote site for evaluation and epidemiological use. Placing AEGIS components in the stems of molecular beacons lowers noise (thus reducing the ‘signal to noise’ ratio) by preventing unwanted cross-reaction with natural DNA that is abundant in natural environmental and human diagnostic samples.

As a further innovation, the diagnostic kit engineers the beacon so that a single target molecule turns over multiple copies of the beacon, allowing it to amplify the signal, increasing the sensitivity of the assay.

Combining these technologies allows the detection of as few as 50 copies of MERS-CoV RNA in the form of a green glow visible to the human eye, significantly stronger than existing point-of-sampling kits. In addition to detecting MERS, it provides accurate results in detecting influenza A and B, severe acute respiratory syndrome (SARS) and human respiratory syncytial virus (RSV); all viruses easily confused with the symptoms generated by MERS.

This new diagnostic device will allow quicker point-of-care testing of infected warfighters allowing for more rapid treatment and troop safety. After analytical comparisons with current CDC kits, the next step will be to gain ‘Emergency Use Authorization.’ This will accelerate the FDA development process, allowing the diagnostic tool to reach the warfighter sooner.

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