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Development of a visible loop mediated isothermal amplification assay for rapid detection of Bacillus anthracis

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Abstract

Distressing effects on animal and human health with lethal progression, being used as bioweapon and shared features with non-pathogenic bacteria demands sensitive, specific, safe, cost effective and rapid detection methods for anthrax causing organisms. Conventional microbiology based diagnostics for anthrax are time consuming and need sophisticated equipment, while molecular diagnostics require less time and labor. The Loop mediated isothermal amplification assay (LAMP) is rapid, sensitive and specific assay and requires no specialized equipment. In the present study, we developed a LAMP assay for rapid as well as specific detection of Bacillus anthracis. The optimized assay produced positive results with the Sterne strain and one field isolate of B. anthracis and, negative results with other bacteria of the same and different genera within 2 h. Sensitivity was 500 fg of total DNA of B. anthracis, which was 100 times more sensitive than conventional PCR. The present study also demonstrated that the simple method of total DNA extraction by repeated boiling and freezing will not adversely affect the LAMP results. In conclusion, the optimized LAMP assay is a promising tool for the specific, sensitive, less time-consuming diagnosis for anthrax causing bacteria and also, for detecting the virulence of suspected B. anthracis cultures.

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... DNA extracted from Formalin Fixed Paraffin Embedded (FFPE) sections was analysed in the DIMES zoonoses laboratory with the attempt of identifying pathogens causing look-alike diseases. A panPox Real-Time PCR was carried out to detect Poxviridae DNA(Luciani et al., 2021) while a Loop-Mediated Isothermal Amplification (LAMP) and a Realtime PCR were performed to detect Bacillus anthracis(Upadhyay et al., 2021;Kędrak-Jabłońska et al., 2018). The positive result to the panPox Realtime PCR was subsequently confirmed by HGC Realtime PCR(Li et al., 2010) that revealed the presence of PPV DNA. ...
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