A Novel Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Yersinia pestis

Abstract

Rapid detection of Yersinia pestis, the causative agent of plague, is essential during field investigations to enable prompt control measures for prevention of the spread of the disease. Affordable, efficient, reliable, and simple detection assays are extremely useful, particularly in plague-endemic regions with limited resources. We developed a loop-mediated isothermal amplification (LAMP) assay that detects Y. pestis within 30 min by simply incubating at 65°C on a dry bath heater. The assay targeted the caf1A gene that is situated on the pMT1 plasmid using six specific primers. Y. pestis presence is visually detected based on the color change in the reactions. For comparison of the assay performance, a real-time LAMP with fluorescent dye detection was conducted on a real-time PCR instrument using the same six primers. Sensitivity assessment showed that the limit of detection (LOD) was 0.2 and 0.03 pg when performed on the dry bath heater and on the real-time PCR instrument, respectively. The assay was 100% specific, having no cross-reactivity with closely related Yersinia spp. and other bacterial species. We tested the LAMP assay on field-collected fleas and showed that it successfully detected Y. pestis with identical results to that of a previously published pentaplex real-time PCR assay. These findings suggest that the relatively inexpensive and simpler LAMP assay could be used to support field investigations, yielding comparable results to more expensive and complex PCR assays.

Development of a quadruplex PCR amplicon next generation sequencing assay for detection and differentiation of Bartonella spp.

“…The inhibiting substances may be present in the analyzed samples and may affect the sensitivity of an assay (Kaneko et al, 2007 ; Nkouawa et al, 2010 ). Previous studies have reported the presence of inhibiting substances from DNAs of mouse liver, spleen, and lung in PCR (Feng et al, 2018 ; Bai et al, 2022 ). Our results also may suggest the presence of inhibiting substances from spleen-derived DNAs that have occurred in the testing.…”