Germplex PCR and massively parallel sequencing: A dynamic duo for detecting bacteria of biodefense and public health...

Biological agents may pose a danger to public health and may be used as bio-warfare tools. Whether a natural outbreak, isolated cases of diseases, or intentional dissemination of these agents, the spread of harmful biological agents can have a significant impact on a country's health and security and the effects can extend beyond borders to even the world population. Thus, it is important to identify pathogens and their potential biological risks and if possible, to differentiate natural outbreaks from those that occur intentionally. Despite the latest technological advancements, there is still a need for highly sensitive and specific methods to detect a broad range of clinically relevant microorganisms and recognized bioterrorism agents in an expeditious manner. A multiplex primer panel (called Germplex) with high sensitivity and specificity was developed to identify select potential biological weapons and other clinically relevant pathogens. The panel targets 34 bacterial species of public health and bioterrorism significance. Germplex is comprised of 52 primer pairs targeting specific genes associated with these pathogens including three general purpose primer pairs for the hypervariable region of the 16S rRNA gene. To address the challenge of accessing samples from certain restricted-access bacteria, 31 synthetic DNA fragments were designed corresponding to part of the respective virulence genes. Human or environmental DNA was added to simulate (or Mock) samples of bacterial DNA plus synthetic DNA fragments to determine Germplex's efficacy. Sequence data were mapped against the reference sequences using a customized pipeline. The Germplex panel identified a selected group of bacteria, at 1000 genomic equivalents (GE). For samples containing different amounts of co-mingled environmental DNA the Germplex panel and subsequent sequencing still effectively detected the targets. The amplification efficiency of Germplex at 5000 GE was comparable to that of shotgun sequencing at 50,000 GE. The Germplex panel is an effective strategy for rapid and accurate detection of biological agents which can be readily transferred to most operational laboratories and can be considered as an approach for fast response in public health crises.