qPCR results from design and partial validation of three novel eDNA qPCR assays for several common North American tick (Arachnida: Ixodida) species
Data files
Jan 08, 2024 version files 33.97 KB
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README.md
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TickeDNA.QPCR.Results.xlsx
Abstract
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.
README: Tick qPCR Results
https://doi.org/10.5061/dryad.q2bvq83s3
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.
Methods
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.