![]() ![]() With automation and miniaturization, microfluidics-based chips can be easily used as a POC platform. Development of quantitative, multiplex platforms is imperative for the specific diagnosis of such pathogens, and these platforms can be easily used as point-of-care (POC) molecular diagnostic tools. Molecular diagnostic techniques (Microarrays, PCR and NGS) are time saving and precise in the detection and discovery of micro-organisms in a given sample. PCR amplification cannot produce a large amount of high-throughput data, but next-generation sequencing (NGS) platforms provide an unbiased-in depth analysis of near-complete genomes. Scientists are encouraged to use nucleic acid amplification tests (NAATs), for real-time detection of DNA and RNA using fluorescent dyes through multiplex polymerase chain reaction (PCR) assays, in which multiple pathogens can be diagnosed in a single run. With different types of immuno-based and chip-based biosensors, biothreat agents are quickly and sensitively detected. Immunochromatographic assays, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of biothreat agents. The amalgamation of EM with cell culture practices greatly contributed in the diagnosis of bacterial and viral pathogens. In addition, bacteria are far too thick for transmission electron cryomicroscopy to allow resolution of structural details. Nonetheless, the technique requires a high amount of sample (~1 mg/ml of virus particles). Advancement in the technology led to the development of transmission electron cryomicroscopy, for which specimens are preserved at −150 ☌ for maintaining the amorphous texture and for avoiding any ice-crystal damage. Further, microscopic techniques required expertise and trained manpower. In addition, sample losses were high during sample preparation (electron microscopy, EM), either due to sample dehydration or charging of biomolecules (bacteria and viruses), which used to result in reduced contrast and performance. Diagnosis was based on the morphological features of bacteria and virus particles, which required large amounts of samples, In the case of viruses, a high viral titer (10 5 to 10 6 virus particles/ml) was needed. ![]() The non-cultivable viruses, like rotaviruses, hepatitis A virus, and Norwalk virus, were also identified through microscopic techniques. Different stains (e.g., gram-stain, Rhodamine, Indian ink) on histopathological specimens differentiated and classified several bacterial strains. Microscopy played a pivotal role in diagnosis of bacterial pathogens and helped to enumerate pathogens in different samples (e.g., blood, urine, sputum, stool). Prior to the development of high-end diagnostic tools, basic microscopy techniques used to be a primary tool in the diagnosis of disease. The categorization depends upon the following criteria: (1) the easy dissemination (person-to-person) of biologically active infectious agents, (2) a major public health concern and causing a high case fatality rate both in humans and/or livestock, and (3) the creation of panic and terror among people. The Center of Disease Control and Prevention classified bioterrorism agents into three major categories: Category A, B and C. The Biological Weapon Convention (BWC) was established in 1972, banning the development, production and stockpiling of microorganisms and toxins that can lead to mass destruction. Keywordsīioterrorism is the purposeful and intentional delivery of bioweapons by non-state actors to harm humans, livestock and agriculture. Therefore, the future prospects of molecular based technique should focus on developing quick, user-friendly, cost-effective and portable devices against biological attacks and surveillance programs. The high-end molecular platforms like NGS and Microarray are time-consuming, costly, and produce huge amount of data. In addition, several point of care (POC) devices have been approved by FDA and commercialized in markets. Different molecular and immunological approaches such as Immunochromatographic and lateral flow assays, Enzyme-linked Immunosorbent assays (ELISA), Biosensors, Isothermal amplification assays, Nucleic acid amplification tests (NAATs), Next Generation Sequencers (NGS), Microarrays and Microfluidics have been used for a long time as detection strategies of the biothreat agents. ![]() The advancement in Molecular techniques has been implicated in the development of sophisticated, high-end diagnostic platform and point-of-care (POC) devices for the detection of biothreat agents. ![]()
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