Analysis of macerated ticks using Boolean logic gating colorimetric isothermal nucleic acid assays for Lyme Borrelia and Ixodes scapularis ticks

Abstract

Lyme disease, one of the most common tickborne diseases, has been rapidly spreading in parallel with the expansion of the range of its tick vector. Better tick surveillance efforts are needed to accurately estimate disease risk and to guide public health and clinical management. We have developed two multiplex loop-mediated isothermal amplification (LAMP) reactions coupled with oligonucleotide strand displacement (OSD) probes to identify the tick host, Ixodes scapularis, and the Lyme disease pathogen, Borrelia burgdorferi, they carry. In each multiplex LAMP-OSD assay the co-presence of two target sequences is computed at the DNA level by linking the two corresponding amplicons and detecting the co-product on colorimetric lateral flow dipsticks. In tests with synthetic DNA, the co-presence of as few as four copies of input DNA could be detected, without producing spurious signals. Most importantly, though, the LAMP-OSD assay is amenable to being carried out directly with macerated tick samples, without any sample preparation. In such field conditions, assays performed robustly and demonstrated 97–100% sensitivity and 100% specificity with both field-collected and lab-raised artificially infected ticks. Such easy-to-use, arthropod and pathogen-specific assays would be well suited to field and near patient use without relying on complex instrumentation or infrastructure.