Methods

Toward a CRISPR-based point-of-care test for tomato brown rugose fruit virus detection
Molecular Biology rugose brown fruit virus

Virus isolates and plant inoculation

The Spanish ToBRFV isolate from Vicar (Almeria, Spain) (Alfaro-Fernández et al. 2020) was provided by the “Laboratorio de Producción y Sanidad Vegetal” (La Mojonera, Almería, Spain). Isolates of the other tobamoviruses used in this study were acquired from the DSMZ collection: tobacco mosaic virus (TMV, PV-1252), tomato mosaic virus (ToMV, PV-0141), pepper mottle mosaic virus (PMMoV, PV-0093), and tobacco mild green mosaic virus (TMGMV, PV-0124). Approximately 50 mg of dried plant tissue were homogenized from each isolate in 2 mL of 30 mM phosphate buffer pH = 8 using a mortar and pestle. Homogenates were used to mechanically inoculate 25-26 days old leaves (4-5-true leaves) of N. benthamiana plants and the first pair of true leaves of 7-10 days-old tomato plants (cultivar M82). For this, the leaves to be inoculated were first dusted with carborundum powder (600 mesh) and then rubbed with the homogenate manually. The plants inoculated were kept separately in a confined greenhouse under controlled conditions set at 16/8 hours photoperiod and 26/22 °C in a day/night cycle. After 10 to 15 days, the leaves that showed obvious symptoms of infection were collected, cut, mixed, and divided into samples of approximately 100 mg. The samples were frozen in liquid nitrogen, ground with a Retsch Mixer Mill MM400 for 1 minute at 30 Hz, and stored at -80 ° C for later analyses.

Column-mediated RNA extraction

Column RNA extraction was performed using a NucleoSpin RNA plant kit (MACHEREY-NAGEL, Germany) following the manufacturer’s instructions. The RNA was checked by running a 1% agarose gel, its concentration measured with a NanoDrop One (ThermoScientific, USA), and adjusted to a working concentration of 10 ng/µL to be used as a template for the RT-LAMP, RT-PCR, and RT-qPCR.

RT-LAMP

Loop-mediated isothermal amplification primers (Table S1) were designed using PrimerExplorer v.5 (https://primerexplorer.jp/e/). RT-LAMP reactions were performed using WarmStart LAMP Kit (NEB, USA) at a final volume of 10 μL. Two sets of primers were designed for amplification of the ToBRFV MP ORF (MP1 and MP2), and an additional set as a positive detection control (PDC) to amplify the 25S ribosomal RNA from solanaceous species. Primers were added at a final concentration of 0.2 μM for F3 and B3, 1.6 μM for FIP and BIP primers, and 0.8 μM for LF and LB primers. The reactions were performed independently for each set of primers (MP1, MP2, and 25S) using 2 μL of input RNA. The amplification was performed at a constant temperature of 62 °C for 25– 45 minutes (50-90 cycles of 30 seconds each) in an Applied Biosystems StepOnePlus Real-Time PCR System (USA) and tracked with a DNA-intercalant green fluorophore provided with the WarmStart LAMP Kit (NEB, USA). The RT-LAMP amplification was set to 45 minutes for experiments illustrated in Figures 2, 3A and 3B. This incubation was reduced to 25 minutes for experiments in Figures 3C, 3D and 4.

Figure 2:Design and assessment of a CRISPR-based testing method for the detection of ToBRFV MP ORF.

A). Representation of the ToBRFV genome and the oligonucleotides used for the detection of the MP ORF sequence (MP1 and MP2). The position of the RT-LAMP primers is represented by the black rectangles (F3, B3, FIP, BIP, LF and LB). The stripped rectangles represent the binding position of the F1c and the B1c halves of the FIP and BIP primers. The blue rectangles represent the location of the gRNAs (gRNA MP1 and gRNA MP2). An additional set of primers was used to amplify the rRNA 25S as PDC (25S, not shown). B) Evaluation of the specificity of the CRISPR-based test MP1, MP2 and 25S targets using a no template control (NTC), a healthy-plant RNA extract (WT) and samples infected with different tobamoviruses (PMMoV, TMGMV, ToMV and TMV) related to ToBRFV, using a fluorescent reporter. Bars represent the average of 3 technical replicates (black dots). C) Evaluation of the specificity using a biotinylated reporter with lateral flow strips and TMV-, ToMV-, TMGMV, PMMoV- and ToBRFV-infected tomato plants (T is the test line, and C the control line).

Figure 3:Comparison of the detection limits of the CaTa28 RT-qPCR and the CRISPR-based tests for the detection of ToBRFV.

Limit of detection of RT-qPCR and CRISPR estimated with serial dilutions of a synthetic RNA transcript fragment of the MP ORF (A, B) or an RNA extract from ToBRFV-infected tomato leaves (C, D). Each RNA dilution was assessed by triplicate. As negative controls, no template control (NTC) and healthy tomato plant (wild type, WT) RNA extract were used. A healthy tomato plant RNA extract was used as diluent in all cases. The dotted lines show the cut-off value.

LbCas12a trans-cleavage assay

LbCas12a trans-cleavage assays were performed using fluorescent and biotinylated reporters, as described by Broughton et al. 2020 and Tsou et al. 2019. When using a fluorescent reporter, 40 nM LbCas12a (EnGen Lba Cas12a, NEB, USA) was pre-incubated with 40 nM of chemically-synthetized gRNA (Synthego, USA, Table S1), and 100 nM of an ssDNA fluorescent (Table S1) reporter for 10 minutes at 37 °C. Then, 2 μL of LAMP product was combined with 18 μL of LbCas12a-gRNA complex and incubated for 10 minutes at 37 °C (20 cycles of 30 seconds each) in an Applied Biosystems StepOnePlus Real-Time PCR System (USA) to detect the resulting fluorescence. When using a biotinylated reporter, the amount of LbCas12a was increased to 100 nM and the gRNA to 125 nm maintaining the pre-incubation time and the 100 nM of the reporter molecule (Table S1). Again, 2 μL of LAMP product was mixed with 18 uL of LbCas12a-gRNA-reporter mixture and incubated for 10 minutes at 37 °C. Finally, 80 uL of 1X HybriDetect assay buffer was added to the reaction mixture, and the lateral flow strip (Milenia HybriDetect - Universal Lateral Flow Assay Kit, Germany) immersed for 2 minutes. Images of the LFA strips were collected using an office scanner and quantified with the ImageJ software.

RT-PCR

The cDNA was prepared using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland) following the manufacturer’s instructions, and AB-783 oligonucleotide priming the 3’-UTR of ToBRFV. The resulting cDNA was diluted in 1/10 with water and used as a template for PCR amplification of a fragment of the MP ORF, by using Phusion Hot Start II High-Fidelity DNA Polymerase (ThermoFisher, USA) and a final concentration of 0.5 µM AB-782 and AB-783 primers (Table S1). The PCR product was assessed by 1% agarose gel electrophoresis and cleaned using GeneClean Turbo Kit (MP biomedicals, USA) silica gel columns.

LbCas12a-mediated restriction reaction and efficiency estimation

A restriction reaction was set containing 250 ng of the same PCR product obtained in the previous section “RT-PCR”, 1 µL 1 µM LbCas12a, 1 uL 1 µM gRNA of each target (MP1, MP2 or 25S), 5 µL 10X NEB2.1 and RNase-free water up to 30 µL. The reaction mixture was incubated for 1 hour at 37 °C, after which the restriction product was resolved in a 2% agarose gel for 45 minutes. The efficiency of each target was estimated using ImageJ for the quantification of the digested and nondigested bands. The value of the digested band was divided by the sum of the non-digested plus the digested and the resultant value multiplied by 100 to obtain the %Efficiency shown in Figure S3.

In vitro transcription

RNA fragments of the ToBRFV MP ORF were synthesized from a PCR product (see “RT-PCR” section) that included the T7pol promoter. One µL of PCR product was employed to produce the transcripts with the T7 RNA polymerase (Promega, USA), setting a 20 µL-reaction for 2 hours at 37 °C. Then, 3 µL of DNAse I (NEB, USA) were added, and the mixture incubated for 15 minutes at 37 °C. The RNA was precipitated by adding water to 100 µL, 10 µL 3M sodium acetate, 250 µL of ethanol, and incubated for 1 hour at −80°C. The RNA was sedimented by centrifugation at 13,000 rpm for 30 minutes at 4 °C, the supernatant was discarded, and finally, the pellet was air-dried and resuspended with 20 µL of RNase-free water. The resulting RNA was checked as described in the “Column-mediated RNA extraction” section.

RT-qPCR

Real-time quantitative PCR was performed using KAPA PROBE FAST Universal One-Step qRT-PCR (Roche, Switzerland) and a specific pair of primers and a probe for amplification and detection of ToBRFV (Table S1). Reactions of 10 µL were carried out using a final concentration of 100 mM oligonucleotides and up to 100 ng of RNA template. A StepOnePlus Real-Time PCR System (Applied Biosystems, USA) thermal cycler was employed following this program: reverse transcription at 42 °C for 5 minutes; denaturation at 95 °C for 3 minutes; 40 cycles of amplification with a denaturation step at 95 °C for 3 seconds and annealing and elongation steps at 60 °C for 30 seconds.

ToBRFV time-course experiment

In total, twelve 5 weeks-old tomato plants cv. M82 (3-4 pairs of leaves approximately) were mechanically inoculated (see above). Three plants per data point (1, 2, 3, 4 days post-inoculation) were sampled, collecting the first pair of new leaves that has been observed to accumulate more virus (van de Vossenberg et al. 2020). Then, the pair of leaves were finely sliced and mixed, after which 100 mg of tissue were introduced into a 2 mL tube with a pair of metal beads for subsequent RNA extraction following the column or the paper strip mediated protocols. These samples were frozen in liquid nitrogen and stored at -80 ° C for later analysis.

Paper strip-mediated RNA isolation

A rapid RNA extraction protocol was performed following the protocol reported by Zou et al. 2017. Briefly, samples were lysed by shaking them manually in 500 µL of lysis buffer (20 mM Tris pH = 8.0, 25 mM NaCl, 2.5 mM EDTA, 0.05% SDS). Next, a home-made cellulose dipstick was introduced three times in the crude extract to retain the nucleic acids and then washed three times in 1.75 mL of wash buffer (10 mM Tris, pH = 8.0, 0.1% Tween-20). After this, the nucleic acids retained to the dipstick were directly eluted into the LAMP mixture.

Article TitleToward a CRISPR-based point-of-care test for tomato brown rugose fruit virus detection

Abstract

Implementing effective monitoring strategies is fundamental to protect crops from pathogens and to ensure the food supply as the world population continues to grow. This is especially important for emergent plant pathogens such as tomato brown rugose fruit virus (ToBRFV), which overcomes the genetic resistance resources used in tomato breeding against tobamoviruses and has become pandemic in less than a decade. Here we report the development of a CRISPR/Cas12a-based test to detect ToBRFV in the laboratory and potentially in a field setting. Using different tobamoviruses to assess specificity, our test showed a clear positive signal for ToBRFV-infected samples, while no cross-reactivity was observed for closely related viruses. Next, we compared the limit of detection of our CRISPR-based test with a reference real-time quantitative PCR test widely used, revealing similar sensitivities for both tests. Finally, to reduce complexity and achieve field-applicability, we used a fast nucleic acid purification step and compared its results side by side with those of a commonly used column-mediated protocol. The new protocol saved time and resources but at the expense of sensitivity. However, it still may be useful to confirm ToBRFV, detection in samples with incipient symptoms of infection. Although there is room for improvement, to our knowledge this is the first field-compatible CRISPR-based test to detect ToBRFV, which combines isothermal amplification with a simplified nucleic acid extraction protocol.


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