Specimens examined
Seven Australian genetic strains (clonal genotypes) of phylloxera were assessed in this project: G1, G4, G7, G19, G20, G30 and G38; genotype numbers follow14. These strains were obtained from live insect colonies maintained by Agriculture Victoria Rutherglen, Victoria. Root aphids, mealybugs and other non-target invertebrates were collected from vineyards for assay specificity testing. Additional aphids, from grass and vegetable host plants, as well as Phylloxeroidea (Adelges spp. and Pineus sp.) from pine trees, were also collected to create a comprehensive Aphidomorpha (Phylloxeroidea/Aphidoidea) species panel to test the specificity of the new LAMP assay.
Phylloxera DNA extractions and molecular variation
DNA was extracted from 100% ethanol preserved whole phylloxera adult, crawler, and eggs (destructive extraction method) using a DNeasy Blood and Tissue extraction kit (Qiagen, Australia), following the manufacturers protocol. The DNA concentrations were quantified using a Qubit 2.0 Flourometer (Invitrogen, Life Technologies, Australia) following the manufacturers protocol and stored at − 20 °C. These samples provided “clean” DNA preparations to use for phylloxera DNA barcoding, to assess Cytochrome Oxidase I (COI) genetic variation between strains, and for development of the LAMP assay.
All specimens, both phylloxera and the non-target invertebrates, were identified morphologically, prior to DNA extraction, followed by molecular identification using standard DNA barcoding methods7,17. DNA sequences from the 5′ region of the COI locus were obtained using LCO1490 / HCO2198 primers19. The thermal cycling conditions consisted of a PCR amplification profile of: 2 min at 94 °C, 40 cycles of at 94 °C for 30 s, 50 °C for 45 s and 72 °C for 45 s, and a final extension of 2 min at 72 °C.
PCR amplicons were sanger sequenced by Macrogen Inc. (Seoul, Korea) and sequences were compared to public databases (NCBI and BOLD), to determine similarity (i.e. > 99% matches) with previously identified reference species. DNA sequences from the laboratory strains of phylloxera were used to develop phylloxera specific LAMP primers in this research.
An additional in-field compatible non-destructive DNA extraction method for “crude” DNA extractions was tested. Intact specimens of phylloxera (egg, crawler, and adult) were processed using a modified HotSHOT protocol “HS6” from20. There are several variations of HotSHOT method which have been used previously to extract DNA from mice (HS4)16 and Zebrafish (HS3)21. The HotSHOT (HS6) protocol has been refined to further improve the efficiency of extracted DNA by combining NaOH and Tris–EDTA buffer in a single step20. In our study twenty microlitres of HotSHOT extraction buffer consisting of 25 mM NaOH + TE buffer, pH 8.0 (Invitrogen , Australia) (1:1 volume) was pipetted into each 8 well strip of LAMP PCR tubes (OptiGene, UK). Phylloxera samples were removed from ethanol and air dried on a paper towel for approx.1 min. Single whole adult, crawler, single and/or multiple eggs were transferred with a toothpick (single use to prevent cross contamination), into each well. Each sample was immersed in HotSHOT buffer with up to six samples processed simultaneously in the portable real-time fluorometer (Genie III, OptiGene, UK). The protocol used the Genie III as an incubator at 95 °C for 5 min followed by > 1 min incubation on ice. The DNA was quantified using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher, Australia) and stored at − 20 °C.
Development of the LAMP assay
LAMP primer design
Phylloxera specific primers were designed from an alignment of publicly available COI (5′-region) DNA sequences (from BOLD, http://www.boldsystems.org), which included (i) target (phylloxera) and (ii) non-target species that were either closely related to phylloxera (i.e. Phylloxeroidea), or aphids (Aphididae) known to utilise grapevines or plant roots as hosts (Supplementary Table 1).
For all primers, the GC content (%), predicted melting temperature (Tm), and potential secondary structure (hairpins or dimers) were analysed using the Integrated DNA Technologies (IDT) online OligoAnalyzer tool (https://sg.idtdna.com/calc/analyzer), using the qPCR parameter sets. Complete sets of LAMP primers were analysed together to detect potential primer dimer interactions using the Thermo Fisher Multiple Primer Analyzer tool (www.thermofisher.com). Potential within-species genetic variation in phylloxera LAMP primers was examined using combined DNA sequences of Australian phylloxera and reference sequences available on BOLD (i.e. the seven laboratory strains, plus 230 × 5′-COI reference sequences on BOLD, accessed May 2018).
A synthetic 221 bp DNA fragment, designed from the complete fragment used for the assay (Fig. 1), was synthesized as a gBlock (Integrated DNA Technologies, Iowa, USA) to provide a reliable LAMP positive control. This fragment consisted of all eight LAMP primer regions (Fig. 1) concatenated together, with the non-primer DNA removed from between primers and replaced a run of “ccc” DNA bases between the primer regions and at the ends of the fragment, to increase the overall GC content of the synthetic fragment.
LAMP primer ratio optimisation
Primer master mix was prepared following published protocols11. Optimisation of the six primer (Table 1) ratio and concentration for the phylloxera LAMP assay was also conducted according to11.
LAMP assay conditions
The phylloxera LAMP assay was performed following the same method as published11. All LAMP assays were run in the Genie III at 65 °C for 25 min followed by an annealing curve analysis from 98 °C to 73 °C with ramping at 0.05 °C/s. The total run time being approximately 35 min.
Comparison of molecular methods for phylloxera identification
Analytical sensitivity of the LAMP assay compared with qPCR
Clean DNA was extracted from phylloxera (3 adult samples were pooled for one DNA extraction) using the destructive method. A fourfold serial dilution of two biological replicates was prepared using ultrapure water. Starting DNA concentrations were quantified using a Qubit 2.0 Flourometer (Invitrogen, Life Technologies, Australia) following the manufacturers protocol. Phylloxera DNA was serially diluted from 1.0 ng/µL to 0.000061 ng/µL (1:1 to 1:16,384). Sensitivity of the LAMP assay was tested using the serially diluted DNA in the Genie III, following the same assay conditions as mentioned above. The time of amplification and anneal derivative temperature was recorded for all samples.
The same serial dilution of DNA extracts was also used in real-time qPCR assay. The primers and probe set (manufactured by Sigma, Australia) and cycling conditions used were as published9. Real-time qPCR was performed in QuantStudio 3 Real time PCR system (Thermo Fisher Scientific, Australia) in a total volume of 25 µL with technical replicates for each dilution. Each reaction mixture included 12.5 µL Platinum Quantitative PCR SuperMix-UDG (Invitrogen, Australia), 0.5 µM of each forward and reverse primers, 0.2 µM Taqman probe, 4 µL of template DNA and made up to 25 µL with RNA-free water. An NTC with 4 µl of water instead of DNA was included in each run to check for reagent contamination. The thermal cycling conditions consisted of a two-step denaturation: 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of amplification in a two-step procedure: 95 °C for 15 s and 60 °C for 1 min. The mean Cq value (cycling quantification value) of the 8 dilutions were recorded for comparison with the time of amplification obtained from the LAMP assay.
Performance of non-destructive DNA samples (HotSHOT HS6)
DNA was non-destructively extracted from whole phylloxera egg, crawler, and adult (n = 3) using the “crude” HotSHOT HS6 protocol, as mentioned above. The extracted DNA was tested across the full range of molecular techniques available for detection of phylloxera: real-time qPCR (using 5 µL and 2 µL DNA per reaction)9, microsatellite genotyping14, DNA barcoding7, as well as the new LAMP assay. Real-time qPCR and DNA barcoding were performed as described above. Microsatellite genotyping was performed following a laboratory protocol (Blacket et al. unpublished) that screens the six phylloxera microsatellite loci used to define Australian genotypes5,14, modified to utilise fluorescently labelled primer tails following22 and a multiplex PCR kit (Qiagen, Australia). Capillary separation of fluorescently labelled microsatellites was performed commercially by AGRF (Melbourne), using LIZ-500 size standards. Genotyping of fsa files was performed using the microsatellite plugin in Geneious R11 (https://www.geneious.com).
Phylloxera specimens (adults and crawlers) were visually examined pre-DNA extraction, with slides prepared from specimens’ post-DNA extraction to confirm retention of key morphological characters. Microscopic slides were prepared following the protocols as published22 and were deposited as reference specimens in the Victorian Agricultural Insect Collection (VAIC) (https://collections.ala.org.au).
Evaluation of gBlock DNA fragment for use as synthetic DNA positive in LAMP assay
To evaluate detection sensitivity a tenfold serial dilution of the gBlock DNA fragment was prepared using ultrapure water (Invitrogen, Australia). Synthetic DNA was serially diluted from ~ 100 million copies down to ~ 10 copies (108 copies to 10 copies). Sensitivity of the LAMP assay was tested using the serially diluted synthetic DNA in the Genie III, following the same assay conditions as mentioned above (run time increased from 25 to 35 min). Following this another LAMP run was conducted to determine the best dilution to be used as synthetic DNA for positive template in LAMP assay. The same fourfold serial dilution (1.0 ng/µL to 0.00098 ng/µL) of clean phylloxera DNA prepared previously was used as template to compare with one hundred thousand copies (105) of synthetic DNA. The amount of phylloxera DNA was then calculated from the amplification time of 105 copies of synthetic DNA.
Source: Ecology - nature.com
