Lakhotia, S. | bioRxiv Science

bioRxiv2023

Novel Loop-Mediated Isothermal Amplification via One Step Strand Displacement Diagnostic of Spotted Fevers

1.Spotted fever rickettsiosis plagues countries around the world. One of the deadliest of this group, Rickettsia rickettsii, responsible for Rocky Mountain spotted fever, is an emerging tickborne illness in North America. The predominant clinical diagnostic is PCR based but does not work until disease has progressed to a severe phase of infection, at which point the outcome of a full recovery is significantly decreased. An alternative, loop mediated isothermal amplification through one-step strand displacement (LAMP-OSD) assay, was developed to improve diagnostic speed and sensitivity. Synthetic dsDNA genes from the 17 kDa surface antigen precursor (AY281069.1) amplified between fifteen minutes to an hour and were detected to concentrations as low as 102 copies/L. This RMSF LAMP-OSD assay shows promise to deliver results in just a few hours and the detection limit is potentially 100 times more sensitive than qPCR based assays.

microbiology↗

bioRxiv2020

High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes

ABSTRACTIsothermal nucleic acid amplification tests (iNAT), such as loop-mediated isothermal amplification (LAMP), are good alternatives to polymerase chain reaction (PCR)-based amplification assays, especially for point-of-care and low resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can generate spurious amplicons, especially in the absence of target sequences, resulting in false positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets, and can accumulate mutations that will lead to inefficient primer binding and thus false negative results. Internally redundant assays targeting different regions of the target sequence can help to reduce such false negatives. Here we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on non-sequence-specific readout into assays that can be visually read using sequence-specific fluorogenic oligonucleotide strand exchange (OSD) probes. We evaluate one-pot operation of both individual and multiplex LAMP-OSD assays and demonstrate detection of SARS-CoV-2 virions in crude human saliva.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗