ABSTRACT
It's been 2 decades since the posting of the anthrax letters in the United States in 2001. This event marked a pivotal point in our history. It highlighted the vulnerability of modern society to acts of bioterrorism and set countries on a course to develop capabilities to pre-empt, prevent, react to, investigate, and recover from acts of bioterrorism. The current COVID-19 pandemic is a stark reminder of the enormity of the impact that the release of a biological agent, natural or otherwise, can have on an immunological naïve society. The purpose of this article is to describe how microbiology is applied in the investigation of bioterrorism, highlighting the modern advances in technology, particularly the DNA technologies, which have assisted this discipline as a forensic practice. © 2023 Elsevier Ltd. All rights reserved.
ABSTRACT
It’s been 2 decades since the posting of the anthrax letters in the United States in 2001. This event marked a pivotal point in our history. It highlighted the vulnerability of modern society to acts of bioterrorism and set countries on a course to develop capabilities to pre-empt, prevent, react to, investigate, and recover from acts of bioterrorism. The current COVID-19 pandemic is a stark reminder of the enormity of the impact that the release of a biological agent, natural or otherwise, can have on an immunological naïve society. The purpose of this article is to describe how microbiology is applied in the investigation of bioterrorism, highlighting the modern advances in technology, particularly the DNA technologies, which have assisted this discipline as a forensic practice.
ABSTRACT
In the Czech Republic, the current pandemic led to over 1.67 million SARS-CoV-2- positive cases since the recording of the first case on 1 March 2020. SARS-CoV-2 genome analysis is an important tool for effective real-time quantitative PCR (RT-qPCR) diagnostics, epidemiology monitoring, as well as vaccination strategy. To date, there is no comprehensive report on the distribution of SARS-CoV-2 genome variants in either the Czech Republic, including Central and Eastern Europe in general, during the first year of pandemic. In this study, we have analysed a representative cohort of SARS-CoV-2 genomes from 229 nasopharyngeal swabs of COVID-19 positive patients collected between March 2020 and February 2021 using validated reference-based sequencing workflow. We document the changing frequency of dominant variants of SARS-CoV-2 (from B.1 -> B.1.1.266 -> B.1.258 -> B.1.1.7) throughout the first year of the pandemic and list specific variants that could impact the diagnostic efficiency RT-qPCR assays. Moreover, our reference-based workflow provided evidence of superinfection in several samples, which may have contributed to one of the highest per capita numbers of COVID-19 cases and deaths during the first year of the pandemic in the Czech Republic.
ABSTRACT
Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer's instructions.