DOI: 10.5061/dryad.q2bvq83s3
title: qPCR results from design and partial validation of three novel eDNA qPCR assays for several common North American tick (Arachnida: Ixodida) species

description: The range expansion of ticks to higher latitudes poses a severe threat to
 human health exposing human populations who had no prior contact with
 ticks to several harmful tick-borne diseases.  Early detection of
 ticks in new areas is critical to help inform the public and medical
 professionals of the dangers associated with tick encounters. 
 Environmental DNA represents a novel survey method that could provide
 reliable records of tick occurrences and timely warnings of their range
 expansions.  In this study, we designed three novel eDNA qPCR
 assays for three common North American tick species (Dermacentor
 variabilis, Amblyomma americanum, and Ixodes scapularis) and tested them
 on samples of grasses collected from grasslands and forests in
 Illinois.  We provide in silico and in vitro validation of all
 three assays, however we were unable to generate any positive detections
 from field samples.  Our lack of eDNA detections likely stems
 from low eDNA deposition rates coupled with rapid degradation in
 grasslands and forests, a problem exacerbated by terrestrial eDNA sampling
 methods that are limited by volume of substrate. We provide
 recommendations for improving sample collection methods to increase
 detection probability in future efforts.  Continued research
 should focus on the viability of eDNA to detect small terrestrial
 invertebrates, like ticks, and it potential as early warning indicator of
 the spread of vector-borne diseases.
description: Plant matter samples containing grasses and leaf litter were
 collected in the fall of 2020, and from May through October of 2021 from
 various sites throughout central and southern Illinois.  Three 250 meter
 linear transects were established at each site and sampled for ticks and
 leaf litter via random selection (without replacement) to reduce bias
 (i.e. one of the three transects was selected at random for each site per
 visit, for a total of three visits on average).  50mL Falcon tubes were
 filled halfway with unpacked plant matter collected at approximately 0m,
 150m, and 250m along the transect to ensure adequate coverage. The leaf
 litter sample tube and a control (blank) 50 mL tube were filled with
 enough CTAB extraction buffer to cover all plant matter, and tightly
 capped. Both tubes were inverted five-ten times (as necessary) to ensure
 all litter was in contact with the buffer. All field
 samples were extracted 30 days post-collection via a modified chloroform:
 isoamyl alcohol method (Renshaw et al., 2015).  All samples and field
 blanks were tested for tick presence via qPCR. All reactions contained
 12.5 µl of TaqMan Environmental Master Mix 2.0, 1 µl of forward and
 reverse primers, 1 µl of probe, 6.5 µl of molecular grade water, and 3 µl
 of DNA.  Three replicates of seven gBlock standards containing target DNA
 from each species were run alongside samples from each plate in order to
 estimate copy number. Concentration of the seven standards ranged from 1
 copy/µl to 1,000,000 copies/µl by a factor of 10.  Thermocycler conditions
 included an initial denaturation step at 95°C for 5 minutes followed by 45
 cycles of 95°C for 30 s, annealing (56.5, 57, and 57.5 depending on
 species) for 30 s, and extension at 72°C for 1 min.
description: # Tick qPCR Results This
 file contains 2 datasets. The first contains all of the Cq values from the
 qPCR plates used to develop limit of detection (LOD) and limit of
 quantitation (LOQ) values for 3 qPCR assays targeting 3 species of ticks
 in Illinois (*Dermacentor variabilis*, *Amblyomma americanum*, and *Ixodes
 scapularis*). The second dataset contains qPCR results from the same three
 qPCR assays on eDNA samples, collected from grasslands and forests in
 Illinois. ## Description of the data and file structure The first dataset
 qPCR results from 3 plates that were used to develop LOD and LOQ values
 for each of the assays. To calculate these values for each assay, we ran
 qPCR plates containing 8 standards with 12 different replicates per
 standard. Each standard contained a known concentration of DNA from a
 gBlock ranging from 1 copy/reaction to 3*10^5 copies/reaction. The
 resulting Cq values were then used to calculate the LOD and LOQ according
 to the procedure outlined in Klymus et al. (2020). In this dataset, we
 have included the name of the standard, the target species, the known copy
 number of that standard (copies/reaction), and the resulting Cq value. The
 Cq value is the cycle number at which the amount of fluorescence exceeds
 the threshold. An "undertermined" in the Cq value indicates that
 this replicate failed to amplify. The Klymus script identified some
 samples as outliers and removed them from calculating the LOD/LOQ. Column
 5 indicates which samples were designated as outliers. This second dataset
 contains both sampling information and qPCR results of field samples. All
 eDNA samples were collected along transects with transects located in a
 randomized block design at sample sites. This dataset contains information
 related to location of transects and sampling date. Also included are the
 qPCR results with Cq values for each of the 3 assays. In addition to the
 results for the eDNA samples, we also included results for our qPCR
 standards and no-template controls. Those samples are given N/A values for
 the sample information and extraction information. qPCR standards contain
 known copy numbers of a gBlock ranging from 1 copy/ul (Standard 7) to
 1*10^6 copies/ul (Standard 1) by a factor of 10. Cq values listed as NA
 indicate no observed amplification.