In 2015, the Bipartisan Commission on Biodefense released its foundational National Blueprint for Biodefense. At the time, the Commission deemed the United States dangerously vulnerable to biological events. By 2023, large-scale biological events have forced some changes to our national and global biodefense, but gaps and inadequacies remain. We must continue to prepare for future pandemics, even as we recover from COVID-19 and respond to the spread of other diseases.

Nanopore sequencing technology has enabled numerous applications for rapidly identifying and characterizing biological threats. Warfighters are being trained to execute these protocols, which will revolutionize biothreat detection in the field. Methods for sample/library preparation are being further simplified/automated for use by non-laboratory trained operators, and bioinformatics software was designed to automatically identify biothreats in real-time. In addition, protocols have been used to monitor for SARS-CoV-2 variants among warfighter populations.

Rapid Acceleration of Diagnostics (RADx) Tech is an NIH-funded program to accelerate development, validation, and commercialization of innovative point-of-care and home-based tests, as well as improvements to clinical laboratory tests, that can directly detect the virus that causes COVID-19 and its variants. The program generated 27 FDA Emergency Use Authorizations for COVID-19 diagnostic tests, including the first over-the-counter test for use at home without prescription, and produced 500 million tests.

U.S. Customs and Border Protection (CBP), a federal border control and management agency, is charged with anticipating, detecting, and disrupting terrorism threats, including biothreats, to protect the people and economy of the United States. CBP has developed specialized resources to counteract biothreats and employs robust outreach efforts for private industry, academic institutions, and external stakeholders to increase biological related material, data, and sensitive technology compliance.

Molecular assays are critical for diagnosing and treating infectious diseases. Mutations in a new variant’s genome may lead to false negative result in a sample containing that variant. Here, we discuss data from wet lab testing of the in silico predictions of SARS-CoV-2 assay failures due to mismatches in assay signatures. We found that the majority of assays with mismatches in primer/probe regions performed without drastic degradation in assay performance. We identified critical nucleotides, positions and types of changes that may impact assay performance. These data can be used to improve in silico predictions by our PCR Signature Erosion Tool (PSET).

Available methods for biological aerosol collection and identification are slow and require manual intervention. In order to achieve rapid, automated, and unmanned identification of aerosolized biothreats, we have developed a portable low-cost prototype that couples a customized microwell aerosol collector with an automated elution module and conducts fully-automated sample preparation and loop-mediated isothermal amplification (LAMP) analysis for rapid and accurate biothreat identification.

Our prototype Biological Malware Detector is a software tool that scans electronic systems for the sequences of biological threats in the same way we currently scan these systems for cyber-threats. This tool could be used for preliminary assessment of the biological threat capabilities and intentions of persons and organizations.

The current infectious disease surveillance system largely rely on disease burden data collected over time within a health care system and then extrapolated to general population. COVID-19 pandemic has led to a rapid development of novel high throughput serological methods that can be very effectively used at population level. I will describe novel serological methods and some examples of their use to evaluate the immune status at population level.

Microbial surface sampling of relatively large surface areas from 1-10 square meters is needed to drive relevant data collection in real-world environments. Successful methods efficiently sample from such areas and deliver final samples with a small liquid volume for use with rapid microbial methods and sequencing techniques. Methods developed by NASA for use in cleanrooms and in space environments will be discussed along with a single-handed sponge-in-bag sampler developed for DoD and commercial use that was demonstrated in a simulated meatpacking environment.

Inadequate rapid testing led to the spread of SARS-CoV-2, the virus responsible for COVID-19, with over 98.7 million confirmed cases and 1.09 million deaths in the US alone as of December 2022. A novel quasi-freestanding epitaxial graphene (QEG) biosensor is presented, capable of electrically transducing biological bindings in seconds. A sensing mechanism unique to QEG, polarization-induced strain, due to the interplay of polarization fields generated by the underlying hydrogen-terminated silicon carbide substrate and the crosslinker, poly-L-lysine (PLL) functionalization. The sensor has demonstrated the detection of as little as 60 copies/mL of SARS-CoV-2 in saliva, nasal, and aerosol samples in seconds.

The analysis of genomic data continues to be at the centerpiece of several detection and characterization efforts. Here we introduce different examples of microbial detection in terrestrial and aquatic environments using a set of complex biological matrices. The implications of this biodetection approach in the development of the biosurveillance and biodefense enterprise will be discussed.