CRISPR-Cas12a assisted recombinase based strand invading isothermal amplification platform designed for targeted detection of Bacillus anthracis Sterne.

Detection of a pathogen is crucial prior to all prophylaxis and post exposure treatment, as it can prevent further disease manifestation. In this study, we have developed a nucleic acid pre-amplification based CRISPR diagnostic for detection and surveillance of Bacillus anthracis Sterne. Strand Invasion Based isothermal Amplification (SIBA) platform and Cas12a (CRISPR endo-nuclease) was used to develop CRISPR-SIBA, a multifaceted diagnostic platform. SIBA was employed as the isothermal pre-amplification platform. CRISPR-Cas12a based collateral trans-cleavage reaction was used to ensure and enhance the specificity of the system. Efficiency of the detection system was evaluated by detecting Bacillus anthracis Sterne in complex wastewater sample backgrounds. Previously reported, Prophage 3, Cya and Pag genes of Bacillus anthracis were used as targets for this assay. The amplification system provided reliable and specific detection readout, with a sensitivity limit of 100 colony forming units in 40 min. The endpoint fluorescence from CRISPR collateral cleavage reactions gave a detection limit of 105 to 106 CFUs. The experiments conducted in this study provide the evidence for SIBA's applicability and compatibility with CRISPR-Cas system and its efficiency to specifically detect Bacillus anthracis Sterne. CRISPR-SIBA can be translated into developing cost-effective diagnostics for pathogens in resource constrained settings.

Recent Advancements and Novel Approaches Contributing to the Present Arsenal of Prophylaxis and Treatment Strategies Against Category A Bacterial Biothreat Agents.

Bacterial pathogens have always been a part of the ecosystem in which we thrive. Some pathogens have caused deadly outbreaks in the past and have been exploited as an agent of threat. Natural hotspots for these biological pathogens are widely distributed throughout the world and hence they remain clinically important. Technological advancement and change in general lifestyle has driven the evolution of these pathogens into more virulent and resistant variants. There has been a growing concern over the development of multidrug-resistant bacterial strains that could be used as bioweapons. This rapid change in pathogens also propels the field of science to develop and innovate new strategies and methodologies which are superior and safer to the existing ones. Some bacterial agents like-Bacillus anthracis, Yersinia pestis, Francisella tularensis and toxins produced by strains of Clostridium botulinum, have been segregated as Category A substances as they pose imminent threat to public health with a history of life threatening and catastrophic disease. This review highlights some encouraging developments and value additions in the current plan of action for protection against these select biothreat bacterial pathogens.