SummaryBurkholderia mallei is the aetiological agent of glanders, a highly contagious and reemerging zoonotic disease. Early diagnosis of glanders is critically important to ensure timely treatment with appropriate antibiotics in humans, and to prevent spread of infection in animals. Molecular detection of B. mallei has always been troublesome because of its genetic similarity with Burkholderia pseudomallei, the causative agent of melioidosis. In present investigation, a set of six B. mallei-specific primers were designed and a simple, rapid, specific and sensitive real-time loopmediated isothermal amplification (LAMP) assay was developed for detection of B. mallei. The LAMP assay could detect as low as 1 pg of B. mallei genomic DNA and 5.5 9 10 3 CFU/ml of B. mallei in spiked human blood. The assay was highly specific for B. mallei as it did not cross-react with other bacterial strains used in the study. The established LAMP assay is field adaptable and can be a better and viable alternative to PCR-based techniques for detection of B. mallei in glanders endemic areas with resource-limited settings.
Summary
Burkholderia mallei, a potential biothreat agent is the aetiological agent of glanders, a zoonotic disease primarily affecting equines. B. mallei shares close genetic proximity with B. pseudomallei, the aetiological agent of melioidosis. Hence, molecular detection of B. mallei and its differentiation from B. pseudomallei has always been challenging. Early diagnosis of glanders is critical for timely treatment in humans and disease containment in animals. In this study a recombinase polymerase amplification‐lateral flow (RPA‐LF) assay has been developed for early and accurate detection of B. mallei. RPA‐LF assay was found to be highly sensitive and detected as low as 10 fg genomic DNA of B. mallei. The assay was highly specific and could differentiate B. mallei and B. pseudomallei. The assay also detected B. mallei in artificially spiked blood, tap water and garden soil. The established assay is simple, rapid and does not require complex instrumentation. The field deployable assay can have better implications in rapid glanders diagnosis and environmental detection of B. mallei over PCR‐based detection tools in glanders endemic areas with limited laboratory resources.
Consistently emerging variants and the life-threatening consequences of SARS-CoV-2 have prompted worldwide concern about human health, necessitating rapid and accurate point-of-care diagnostics to limit the spread of COVID-19. Still, However, the availability of such diagnostics for COVID-19 remains a major rate-limiting factor in containing the outbreaks. Apart from the conventional reverse transcription polymerase chain reaction, loop-mediated isothermal amplification-based (LAMP) assays have emerged as rapid and efficient systems to detect COVID-19. The present study aims to develop RT-LAMP-based assay system for detecting multiple targets in N, ORF1ab, E, and S genes of the SARS-CoV-2 genome, where the end-products were quantified using spectrophotometry, paper-based lateral-flow devices, and electrochemical sensors. The spectrophotometric method shows a LOD of 10 agµL−1 for N, ORF1ab, E genes and 100 agµL−1 for S gene in SARS-CoV-2. The developed lateral-flow devices showed an LOD of 10 agµL−1 for all four gene targets in SARS-CoV-2. An electrochemical sensor developed for N-gene showed an LOD and E-strip sensitivity of log 1.79 ± 0.427 pgµL−1 and log 0.067 µA/pg µL−1/mm2, respectively. The developed assay systems were validated with the clinical samples from COVID-19 outbreaks in 2020 and 2021. This multigene target approach can effectively detect emerging COVID-19 variants using combination of various analytical techniques at testing facilities and in point-of-care settings.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.