DNA Extraction
Animal tissues were harvested from specimens (Northern African python, Burmese python, boa constrictor, and rainbow boa) previously collected during research projects conducted by the University of Florida (UF IACUC protocol number 202200000027; FWC EXOT-23-63a; FWC EXOT-23-83) or from specimens donated by the Florida Fish and Wildlife Conservation Commission acquired through invasive species removal efforts.
Muscle tissue (100 mg) was excised from each specimen and transferred to a 1.5 ml microcentrifuge tube. Total DNA was extracted using the DNeasy® Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions with slight modifications. In the initial lysis step, tissue was not macerated, it was left intact and allowed to lyse over a 24-hour period at 56 °C. Final eluate had DNA concentration and purity measured using a NanoDropLite spectrophotometer (ThermoFisher) and was diluted to 25 ng/µl for running PCR assays.
PCR Assay Design
The gene and region selected for assay design was the cytochromec oxidase subunit I (COI) barcoding region (5’-half) due to high levels of variability among species (and even among populations; Kundu et al. 2020, Liu et al. 2020) allowing for easy development of species-specific assays without cross-amplification. Template obtained from B. constrictor , E. cenchria , P. sebae , andP. bivittatus , was screened in the initial assay using the universal COI primers LCO1490 (forward) and HCO2198 (reverse) from Folmer et al. (1994). Reactions were run in volumes of 25 µl and were comprised of 5X GoTaq Flexi Buffer (Promega, Madison, Wisconsin, USA), 25 mM MgCl2, 200 μM dNTPs, 0.5 μM of forward and reverse primer, 2% PVP-40, 1 U GoTaq Flexi DNA polymerase (Promega, Madison, Wisconsin, USA), and 2 µl of DNA template with sterile water to increase the final reaction volume to 25 µl. Utility of these primers in snakes was previously unknown so insect DNA extract using the same protocol as that for snakes from the planthopper Pelitropis rotulata was used as a positive control and molecular grade water was used as a non-template control. Thermal cycling conditions were as follows; initial denaturation at 95 °C for 2 min. followed by 35 cycles of denaturation at 95 °C for 30 sec., annealing for 30 sec. and extension at 72 °C, followed by a final extension at 72 °C for 5 min (Table 1). Products obtained from PCR reactions were run on 1.5% agarose gel stained with GelRed (Biotium) and amplicons of the correct size, relative to the positive control, were sent for Sanger sequencing (Eurofins Genomics, Louisville, Kentucky, U.S.A.).
Sequence data was assembled using DNA Baser (Version 4.36) (Heracle BioSoft SRL, Pitesti, Romania) and aligned using ClustalW as part of the MEGA7 package (Kumar et al. 2016). Sections of 100 bps displaying variability among the four species included in this study were selected and uploaded to OligoArchitectTM Online (Sigma-Aldrich) using the “Dual-Labeled Probe” tab. Each resulting assay was subsequently purchased as a TaqMan® MGB (minor grove binder) probe with a 5’ FAM label and 3’ nonfluorescent quencher (NFQ) for optimization along with corresponding primers. Each assay was screened against its corresponding snake species with species-specific primers only by standard PCR using a gradient to establish optimal annealing temperatures for the assay. For the gradient PCR, reactions were performed using the same concentrations as the initial PCR assays listed above with a gradient of 50 °C to 60 °C. Each resultant assay was labeled according to abbreviated common names of the snake species for ease of labeling and presentation; B. constrictor specific assay = BC, E. cenchria specific assay = RB, P. sebae specific assay = NAP and P. bivittatus specific assay = BP.
qPCR and dPCR optimization
All assays designed were subsequently screened against the original template used to generate sequences that resulted in the corresponding assays. Each assay specific to a snake species was screened in triplicate against template for the same snake species (representing positive controls) and also screened in triplicate against the other three snake species template (negative controls) to ensure no cross-amplification occurs. All assays were run on a QuantStudio 6 Flex qPCR system (Applied Biosystems by Thermo Fisher Scientific). Reactions were performed in volumes of 20 µl and comprised of 10 µl of TaqMan Universal Master Mix II with UNG, 10 µM for each oligonucleotide (forward primer, reverse primer, and probe), 10% polyvinylpyrrolidone (PVP-40), 1 µl of DNA template with sterile dH2O added to reach final volume (20 µl). Thermal cycling conditions for qPCR assays were as follows; initial hold at 50 °C for 2 min, initial denaturation at 95 °C for 10 min. followed by 35 cycles of denaturation at 95 °C for 15 sec. and annealing/extension at 58 °C for 1 min.
Amplicons from the gradient PCR for each snake species were cloned using the pGEM-T Easy Vector kit (Promega) following the manufacturer’s instructions. The cloned vectors were then transformed into NEB Turbo Competent E. coli (New England BioLabs) and plated on Lysogeny broth (LB) plates containing 100 mg/mL of Ampicillan. Plates were incubated overnight and transformed colonies were screened for the clones with correct inserts using M13F/M13R primers. Clones with an insert of the correct size were incubated at 37 °C overnight on a shaker with 250 rpm in 20 ml of LB broth containing 100 mg/ml of Ampicillan. Finally, plasmids were extracted using a QIAprep Spin Miniprep Kit (Qiagen) per the manufacturer’s instructions and sent for Sanger sequencing (Eurofins Genomics) to confirm identity of the inserts. Plasmid eluate was subsequently diluted to 107copies/µl followed by a serial dilution to 101copies/µl.
Serially diluted plasmids for NAP were subsequently run with corresponding assay on the QuantStudio™ Absolute Q™ Digital PCR System (dPCR) to establish optimal dilution concentration. In addition, eluate from the extraction protocol from raw tissue for NAP was diluted to 25 ng/µl, then subsequently serially diluted (10:1) three times and screened with the corresponding assay to determine optimal concentration for total DNA samples. Optimal concentration was determined only using NAP because the same target is being evaluated across taxa, so with standardized concentrations for both samples and plasmids, optimal concentrations can be determined and extrapolated to the other species.