Computational protocol: Molecular methods to detect Spodoptera frugiperda in Ghana, and implications for monitoring the spread of invasive species in developing countries

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Protocol publication

[…] DNA samples were stored at −20 °C for at least 24 hours before being further processed. A fragment of the abdomen of each specimen was air-dried for 5 minutes, then rinsed with 50 µl sterile molecular grade H2O (ThermoFisher Scientific, UK) to rehydrate the sample and to dilute residual ethanol. Excess water was removed, and DNA templates for PCR amplification were obtained by adding 20 µl of microLYSIS®-PLUS (MLP; Microzone Ltd., UK) to the dried material. The suspension was macerated with a sterile micropestle (VWR International Ltd., UK) to facilitate the disruption of the exoskeleton and tissues of the samples. DNA was then liberated into the MLP by placing the sample tubes in a thermal cycler and subjecting to the heat profile recommended by the manufacturer, for difficult samples.PCR reactions were carried out using a Hybaid PCR Express thermal cycler in heated-lid mode. Amplifications were carried out in 0.5 ml microcentrifuge tubes in 20 µl reactions containing: 1 µl MLP DNA extract; Primers LCO1490 and HCO2198 (5′-GGTCAACAAATCATAAAGATATTGG-3′ and 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′, respectively) each at 150 nM; and 10 μl of MegaMix-Royal (Microzone Ltd, UK) mastermix solution, containing optimised mixture of Taq polymerase in 2 × Enhancing Buffer (6 mM MgCl2), with 400 μM dNTPs and blue MiZN loading dye. Reactions were made up to a final volume of 20 μl sterile molecular grade H2O. PCR reactions were preincubated for 5 min at 95 °C followed by 39 cycles of: 30 s at 94 °C; 30 s at 51 °C; 75 s at 72 °C. Samples were finally incubated for 10 min at 72 °C followed by chilling at 10 °C. In accordance with our standard practice, a ‘no DNA’ negative control (components as above but containing 1 µl sterile H2O instead of DNA) was included with each set of reactions.Where necessary, a second round of amplification (i.e. ‘reamplification’) was undertaken as follows: 1 µl of each of the above PCR products was used as template. The reaction was carried out under the same conditions, with the exception of the number of cycles, which was reduced to 30. In such cases, a fresh ‘no DNA’ negative control was prepared as described previously but an additional negative control was prepared using 1 µl of the first round ‘no DNA’ negative control reaction mix for that reaction set. Aliquots (4 µl) of each PCR product were used for agarose gel electrophoresis with 1.5% (w/v) Hi-Pure Low EEO agarose (BioGene Ltd, UK) in 0.5x TBE (Severn Biotech Ltd, UK) running buffer, containing 5 µl SafeView nucleic acid stain (NBS Biologicals Ltd., UK) for 100 ml of 0.5X TBE, and with 4 µl 100 bp size marker (ThermoFisher Scientific, UK). PCR products of the expected size (ca. 650 bp; see Supplementary Figures  and [It may be noted, also, from Supplementary Figures  and that there was no visible amplification of first and/or second round ‘no DNA’ negative controls, thereby showing that any positive reactions obtained were genuine and not artefactual or contaminant in nature]) were purified using microCLEAN purification solution (Microzone Ltd., UK) in accordance with the manufacturer’s instructions. Purified products were resuspended in 15 µl sterile molecular grade H2O.Sequencing of PCR products was undertaken using a thermal cycler (MWG Primus, Germany) in heated-lid mode with BigDye® Terminator v3.1 cycle sequencing kit (ThermoFisher Scientific, UK). Sequencing reactions contained the following, in 0.5 ml microcentrifuge tubes: 2.68 µl of template DNA prepared as above; Primer HCO2198 at 320 nM; 5x BigDye® Terminator Sequencing Buffer; BigDye® Terminator. The sequencing reactions were preincubated for 1 min at 96 °C followed by 25 cycles of: 20 s at 96 °C; 10 s at 50 °C; 4 min at 60 °C. Samples were finally chilled at 10 °C. Excess unincorporated dye-terminators were removed using DyeEx® 2.0 spin columns (Qiagen, UK) according to the manufacturer’s recommendations, with the eluted purified sequencing reaction products being resuspended in 16 µl of Hi-Di TM formamide (ThermoFisher Scientific, UK) prior to automated capillary electrophoresis and sequence reading on an ABI 3130 Genetic Analyser (ThermoFisher Scientific, UK). Sequences obtained after a second round PCR ‘reamplification’ were of as good quality as those obtained from a single round PCR. Samples were only considered to be positive for FAW (or, indeed, B. fusca) if they gave a good quality sequence – i.e. appearance of a band was not sufficient. This enabled us to ensure that our results were not artefactual, contaminant or chimaeric. PCR success rates were at 60–71% (i.e. nine sequences obtained from 13 samples from Brong Ahafo [69% successful]; 3/5 from Northern Region [60%]; 5/7 from Volta Region [71%]). Sequences were aligned using the multiple sequence alignment plug-in CLUSTALW in MEGA6. Sequences obtained in the present study were compared with authenticated sequences obtained from the Barcoding of Life Data system (BOLD; http://www.boldsystems.org/ ) and additional sequences from the GenBank® data base (http://www.ncbi.nlm.nih.gov/genbank/). Alignment used the default parameters of CLUSTALW and MUSCLE, and these were then optimized manually in the MEGA6 program. […]

Pipeline specifications

Software tools Clustal W, MEGA, MUSCLE
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Spodoptera frugiperda, Caenorhabditis elegans