Validation of droplet digital PCR for the detection and absolute quantification of Borrelia DNA in Ixodes scapularis ticks

Abstract

We evaluated the QX200 Droplet Digital PCR (ddPCR™, Bio-Rad) system and protocols for the detection of the tick-borne pathogens Borrelia burgdorferi and Borrelia miyamotoi in Ixodes scapularis nymphs and adults collected from North Truro, Massachusetts. Preliminary screening by nested PCR determined positive infection levels of 60% for B. burgdorferi in these ticks. To investigate the utility of ddPCR as a screening tool and to calculate the absolute number of bacterial genome copies in an infected tick, we adapted previously reported TaqMan®-based qPCR assays for ddPCR. ddPCR proved to be a reliable means for detection and absolute quantification of control bacterial DNA with precision as low as ten spirochetes in an individual sample. Application of this method revealed the average carriage level of B. burgdorferi in infected I. scapularis nymphs to be 2291 spirochetes per nymph (range: 230-5268 spirochetes) and 51 179 spirochetes on average in infected adults (range: 5647-115 797). No ticks naturally infected with B. miyamotoi were detected. The ddPCR protocols were at least as sensitive to conventional qPCR assays but required fewer overall reactions and are potentially less subject to inhibition. Moreover, the approach can provide insight on carriage levels of parasites within vectors.

Detecting the Lyme Disease Spirochete, <em>Borrelia Burgdorferi</em>, in Ticks Using Nested PCR

“…Only recently have protocols been optimized for higher yield 35 . Meanwhile, the gut of an infected adult tick can harbor on average anywhere from 2,000 to over 50,000 Borrelia 37 38 39 , which is solidly within the detection range of nPCR. Thus, the protocol is well suited to tick surveillance efforts.…”

“…Moreover, the technique has been successfully applied in multiplex reactions to simultaneously detect a mammalian host gene 44 and other tick-borne pathogens 45 , which is not as readily achievable with nPCR. Other design modifications include droplet digital PCR (ddPCR) technology, which can quantify DNA without the use of a standard curve 39 48 . The lower detection limit of this technique for Borrelia is likewise around ten spirochetes/sample 39 .…”

“…Other design modifications include droplet digital PCR (ddPCR) technology, which can quantify DNA without the use of a standard curve 39 48 . The lower detection limit of this technique for Borrelia is likewise around ten spirochetes/sample 39 . Compared to nPCR, these approaches also have the advantage of reduced potential for contamination, as they only require a single amplification protocol.…”

Application of droplet digital PCR to detect the pathogens of infectious diseases

“…However, it is still an available test to confirm Lyme arthritis for its higher sensitivity to detect ospA DNA in synovial fluid [ 36 , 37 ]. Even though there is no reported application of ddPCR to the samples of patients with Lyme disease; recently, the potential advantages of ddPCR to detect the pathogens of Lyme disease in ticks were reported [ 38 ].…”

Molecular diagnostics in the clinical microbiology laboratory

“…Their results showed an increased sensitivity for the detection of M. tuberculosis DNA with ddPCR over qPCR and concluded that ddPCR might potentially be used as a non-invasive, rapid and highly sensitive diagnostics tool for the detection of both pulmonary and extrapulmonary tuberculosis. In another study by King et al 34 the authors used ddPCR for the detection and absolute quantification of Borrelia burgdorferi DNA in adult patients and Ixodes scapularis ticks. They concluded that ddPCR was as sensitive as a qPCR assay, but had some advantages over it, like fewer overall reactions and decreased sensitivity to PCR inhibitors.…”