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bioRxiv · 10.1101/2020.06.16.154336

Development of a sensitive molecular diagnostic assay for detecting Borrelia burgdorferi DNA from blood of Lyme disease patients by digital PCR.

Das, S.·Hammond_McKibben, D.·Guralski, D.·Lobo, S.·Fiedler, P. N.
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Abstract

Lyme disease patients would benefit greatly from a timely, sensitive and specific molecular diagnostic test that can detect the causal agent, Borrelia burgdorferi, at the onset of symptoms. Currently available diagnostic methods recommended by the Centers for Disease Control and Prevention for Lyme disease, involve indirect serological tests that rely on the detection of a host-antibody response which often takes more than three weeks to develop. This results in non-detection of many genuine cases on a timely basis, preventing complete cure. In this study we have developed a digital PCR (polymerase chain reaction) assay that detects Lyme disease on clinical presentation at twice the sensitivity of the currently available diagnostic methods, using a cohort of patient samples collected from the Lyme disease endemic state of Connecticut, USA in 2016-2018. Digital PCR technology was chosen as it is more advanced and sensitive than other PCR techniques in detecting rare targets and the lower limit of detection of this diagnostic assay was found to be three genome copies of B. burgdorferi. The paucity of spirochetes in the bloodstream of Lyme disease patients that hinders the clinical adoption of PCR-based diagnostic tests, was overcome by using a comparatively larger sample volume, pre-analytical processing of blood samples and a pre-amplification step to enrich for B. burgdorferi-specific gene targets before using the digital PCR technology to analyze patient samples. Pre-analytical processing of blood samples from acute patients revealed that the best sample type for Lyme disease detection is platelet-rich plasma and not whole blood. If detected on time, Lyme disease can be cured completely limiting the overuse of antibiotics and associated morbidities.

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Das, S., Hammond_McKibben, D., Guralski, D., Lobo, S., Fiedler, P. N.. 2020-06-16. Development of a sensitive molecular diagnostic assay for detecting Borrelia burgdorferi DNA from blood of Lyme disease patients by digital PCR.. https://doi.org/10.1101/2020.06.16.154336

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