A loop-mediated isothermal amplification assay for the plant-parasitic nematode Aphelenchoides besseyi in rice seedlings

Besides the target species A. besseyi, nematode species that are commonly encountered at the Taiwan boarder quarantine inspections were also included in this study as none targets. A total of 18 nematode samples were used in this study. All nematodes were identified morphologically with microscopes and molecularly confirmed by analyzing the ribosomal DNA sequences (Powers, 2004). Among them, four were intercepted at the custom border, extracted from chopped Black pine (Pinus thunbergii), pine (Pinus spp.) and peat moss. The samples were extracted by using modified Baermann funnel method for 24 hr and were stored in dH₂O at 4°C prior to DNA extraction. The other 14 samples were collected from seven locations in Taiwan, originally extracted from leaf of rice (Oryza sativa), nest fern (Asplenium spp.), or strawberry (Fragaria  ×  ananassa Duch) through modified Baermann funnel method. The sample names, code, origin of the sample and the plant or materials that it was associated with were listed in Table 1. The field-collected samples were sub-cultured every 14 days on slant cultures in test tubes. Aphelenchoides species and B. xylophilus were maintained on Alternaria citri that had been growing on potato dextrose agar (PDA) containing 500 ppm chloramphenicol at 24°C for 5~8 days (Hsieh et al., 2012). Prior to inoculation or DNA extraction, nematodes were harvested from the slant tube by rinsing the agar surface twice with sterilized ddH2O.

Rice seedlings for nematode inoculation were prepared following the standard producing procedure of seedlings producers in Taiwan. Seeds of Taoyuan No. 3 rice (Oryza sativa) were first sterilized in 55°C hot water for 10 min, then soaked in dH₂O at 37°C for two days, and finally placed in autoclave-sterilized soil for three days in the dark to induce germination. Later on, the seedlings were maintained under the day/night regime of 12 hr/12 hr at 28°C for seven days. A. besseyi was directly inoculated onto the seedlings by placing the adult individuals on seedlings with picking needles. For each seedling, 0, 1, 5, 10, 20 or 40 nematodes were inoculated. Total DNA of the inoculated rice were extracted on the same day of inoculation. Three repeats were conducted for each treatment.

Single nematode crude total DNA was prepared after surface-sterilizing the target nematode in 1% NaClO for 1 min, and then rinsed with dH₂O for 1 min. The nematode was then transferred into 20 µl 0.25 M NaOH in 0.2 ml tubes, and incubated for 5 hr at room temperature. The lysate was then heated at 95°C for 3 min, and then 10 μl 0.5 M Tris-HCl buffer, 4 μl 1.25 M HCl and 5 μl 2% Triton X-100 were added in a sequential order into the lysate before the secondary heating at 95°C for 3 min (Floyd et al., 2002). For rice seedlings, plant tissues were first crushed and homogenized by pestle in mortar with liquid nitrogen, and the total DNA was extracted using PowerSoil® DNA Isolation Kit (Mo Bio Laboratories Inc., CA, USA) following the manufacturer’s recommendation. Final DNA products were all stored at −20°C prior to use.

The target gene for LAMP primers was chosen after analyzing all the available sequences of mitochondria and ribosome genes of A. besseyi and its closely related nematode species in the GeneBank database. Multiple sequences alignments were performed using Geneious 9.1.6 software (Biomatters Inc., CA, USA) and the mitochondria COI gene was finally selected as primer target due to its relatively high sequence variation among the Aphelenchoides species. The LAMP primers were designed using PrimerExplorer v.5 software (Eiken chemical Co., Ltd, Tokyo, Japan). Four sets of primers, AB-ID14, AB-ID30, AB-ID37 and AB-ID40, were used for further testing. Among them, the AB-ID37 set contains four primers that attached to multiple sites of the mitochondria COI gene accordingly (Table 2), and all primers in the set had low sequence similarity to other closely related Aphelenchoides species (Fig. 1). All LAMP PCR reactions were conducted in 25-μl reactions. LAMP reaction mixes were prepared using Loopamp DNA Amplification Kit (Eiken chemical Co. Ltd, Tokyo, Japan) as suggested by the manufacturer. The LAMP mix contained distilled water 7 μl, 2× reaction mix 12.5 μl, Primer Mix (FIP 40 pmol, BIP 40 pmol, LF 20 pmol, LB20 pmol, F3 5 pmol, and B3 5 pmol) 2.5 μl, Bst DNA Polymerase 1 μl and 2 μl template. Reactions were set for incubation at 63°C and for the time required for each examination.

Figure 1:

To further understand the developed LAMP assay application potential, a comparison to the prior published detection method was conducted, using the A. besseyi specific PCR primer pair AbF5/AbR5 (Devran et al., 2017) (Table 2). The PCR was conducted in 10-μl reactions. The PCR mix contained 5.1 μl ddH₂O, 1 μl 0.5 M Tris buffer (pH 8.3), 1 μl 25 mM MgCl₂, 0.5 μl 10 g/L BSA, 0.5 μl 2.5 mM dNTP, 0.4 μl 10 mM primer pair, 0.1 μl Taq polymerase (Bioman Scientific Co. Ltd., Taiwan) and 1 μl template DNA. PCR reactions were carried with the following conditions: 94°C 3 min for initial denaturation, 35 cycles with denaturation at 94°C for 30 sec, annealing at 56°C 30 sec, extension at 72°C for 60 sec and final extension at 72°C for 7 min (Devran et al., 2017). Subsequently, 5 μl PCR products were analyzed through electrophoresis in 1.5% agarose, stained with EtBr and imaged with UV light.

To determine the sensitivity of the LAMP assay, serial dilutions of single nematode crude total DNA and the target gene cloned plasmids in sterile ddH₂O were used. The cloned plasmids were prepared using pGEM-T Easy Vector System (Promega, Madison, WI, US). LAMP reaction mixes were prepared using Loopamp DNA Amplification Kit (Eiken chemical Co. Ltd, Tokyo, Japan) as suggested by the manufacturer. The LAMP reaction was conducted in the RealTime Turbidimeter LA-500 (Eiken chemical Co. Ltd, Tokyo, Japan), and the turbidity of each reaction was recorded after each cycle. The specificity of the LAMP assay was tested using crude total DNA of nine above-ground plant-parasitic nematodes species (Table 1). In order to evaluate the application potential of the assay, detection ability of nematode-inoculated rice seedlings prepared as previously described were also examined. Amplification results were analyzed through multiple visualization methods.

Three visualization methods were used and compared for the LAMP assay: gel electrophoresis, hydroxy naphthol blue (HNB) staining and LFD assay. For gel electrophoresis analysis, 30 min were run at 110 V on 2% agarose gels (Bioman Scientific Co. LTD., Taiwan) in an Owl separation system (Thermo Fisher Scientific Inc., USA), then stained in EtBr (Bioshop Canada Inc., Canada) for 10 min, and the electrophoresis result was analyzed in the Gel Doc EZ imager (Bio-rad Laboratories, Hercules, CA, USA) under UV light. HNB (Dojindo Labratories Co. Ltd, Japan) was added at the concentration of 120 μM in each LAMP reaction prior to each reaction. The positive reactions are indicated by a color change from violet to sky-blue (Goto et al., 2009). Custom lateral flow dipstick (LFD) assay was performed using DNA hybridization and immunoprecipitation to specifically detect LAMP reaction products. An oligonucleotide probe labeled with FITC at the 5’ end (Table 2) was designed on COI gene of mtDNA between the F2 and B2 region (Fig. 1). After biotin labeled LAMP reaction, 20 pmol of the oligonucleotide probe was added to the amplification products and kept at 63°C for 10 min for hybridization. Then, 10 μl of the hybridized products was transferred into 150 μl assay buffer (Milenia Biotec GmbH, Giessen, Germany), and the reaction results was analyzed from a customized dipstick after a 5 min reaction time in the mixture.

The AB-ID37 set performed best in the 90 min long efficiency tests; Only 30 min was needed to raise its amplicon mix turbidity above the detection level; whereas 42, 47 and 53 min were needed for AB-ID40, AB-ID30 and AB-ID14, respectively (Fig. 2). Therefore, AB-ID37 was chosen for further exam and optimization.

Figure 2:

All six A. besseyi rice isolates from different geographic locations in Taiwan were successfully detected (Fig. 3A). By contrast, no PCR amplification was observed from nest fern isolates of A. besseyi or other common above-ground plant-parasitic species (Fig. 3A, B). The template with 10⁶ copies/μl had sufficient amplification for turbidity detection after 35 min of reaction. When the reaction time was extended to 90 min, similar to the reaction time of a traditional PCR assay, even the plasmids concentration as low as 10³ copies/μl could be detected (Fig. 4A). The 10^-1 dilution of the crude DNA of single adult nematode was also successfully detected with the assay (Fig. 4B).

Figure 3:

Figure 4:

In the specificity test of primer pair AbF5/AbR5, among the 14 Aphelenchoides samples, all rice and nest fern A. bessyi isolates were detected (Fig. 5A). The sensitivity of the PCR assay is 10³ copies/μl. It was capable of detecting the single adult nematode crude total DNA or its 1/10 dilution (Fig. 5B).

Figure 5:

From a 120-minutes long turbidity recording of AB-ID37 assay reaction, 65 min was shown to be required for the detection power of 10 nematodes per seedling (Fig. 6), and the detection power was extended to 5 nematode per seedling when the reaction time was increased to 85 min (Fig. 6). Gel electrophoresis images, HNB staining results and LFD results from 90 min of AB-ID37 assay reaction were consistent with the turbidity observation (Fig. 7). The detection result is reliable when there are more than 5 nematode per seedling. Compare to electrophoresis, the visualization with LFD saved 15 min of the result analysis time.

Figure 6:

Figure 7: