Recombinase Polymerase Amplification assays for detection of the major tropical root-knot nematodes

Abstract Detection of root-knot nematodes (RKN) in soil and plant samples is crucial to prevent its spread and select effective control measures. In this study, Recombinase Polymerase Amplification (RPA) assays using lateral flow dipsticks (LF-RPA) and real-time fluorescence detection (real-time RPA) were developed to detect the RKN species from tropical complex using a group-specific primer-probe set and Meloidogyne javanica using a species-specific primer-probe set. The results of the real time RPA assays in series of crude nematode extracts showed reliable detection within 16 min with a sensitivity of 1/100 of a second-stage juvenile in a reaction tube. The results of the LF-RPA assays showed reliable detection within 30 min with a sensitivity of 1/20 to 1/100 of a second-stage juvenile and 1/10 of a female in a reaction tube. Real-time RPA and LF-RPA assays are highly specific and can identify their target DNA in mixtures with other nematodes and plant tissues. LF-RPA assay has great potential for diagnosing RKN in the lab, field or in areas with a minimal laboratory infrastructure.

The root-knot nematodes (RKN) of the genus Meloidogyne represent one of the most damaging and agriculturally important groups of plant-parasitic nematodes. Meloidogyne incognita, M. javanica and M. arenaria are three major tropical RKN species, which are globally distributed and polyphagous pests of many agricultural crops. These nematodes together with several other species are commonly referred to the tropical RKN complex (Álvarez-Ortega et al., 2019).
Early and rapid detection of RKN in soil and plant roots is important for the application of effective control measures and prevention of spread of these agricultural pests. Currently, the reliable diagnosis of RKN is based on molecular methods using sequencing of nuclear ribosomal RNA and mitochondrial genes, PCR with specific primers and other techniques (Subbotin et al., 2021). Recombinase Polymerase Amplification (RPA) is a relatively new isothermal methodology for amplifying DNA and uses a highly efficient displacement polymerase that amplifies a few copies of target nucleic acid in several minutes at a constant temperature (37-42°C) (Daher et al., 2016;James and Macdonald, 2015;Piepenburg et al., 2006). RPA has several advantages over PCRbased methods of plant-parasitic nematode detection: (i) does not require thermal cycling and can be used in areas with minimal laboratory infrastructure and run by personals with minimal technical experience; (ii) in 10 or more times sensitive than PCR; (iii) does not require DNA extraction in sample processing; (iv) amplicons may be detected at endpoint or in real-time during 8 to 30 min. Real-time RPA assay with using fluorescent probes in real time for detection of plantparasitic nematodes was first designed and published by Subbotin (2019) for M. enterolobii. Recently, RPA assays with a gel visualization of amplicons for species diagnostics of M. javanica, M. arenaria, M. incognita and M. enterolobii have also been developed by Ju et al. (2019). Chi et al. (2020) designed a rapid RPA assay for visual detection of M. javanica using SYBR Green and lateral flow dipsticks (LF-RPA) at endpoint. RPA assays for detection of the northern RKN, M. hapla were also developed by Song et al. (2021) and Subbotin and Burbridge (2021).
The goal of this study was to develop real-time RPA and LF-RPA assays with group-specific primersprobe sets for the detection of the major RKN belonging to the tropical RKN complex (M. arenaria, M. floridensis, M. hispanica, M. incognita, M. javanica and M. paranaensis) and species-specific primersprobe set the detection of M. javanica only.

Nematode samples
Twenty six isolates of M. javanica, seven isolates of M. arenaria, six isolates of M. incognita, two isolates of M. floridensis and by one isolate of M. hispanica and M. paranaensis were used for RPA assay development and validation. DNA of several other RKN, M. artiellia, M. baetica, M. christiei, M. enterelobii, M. hapla, M. haplanaria, M. naasi and M. nataliei were also used in specificity experiments (Table 1). Second stage juveniles (J2s), eggs and females were extracted from root or soil samples. The nad5 gene was amplified and then sequenced from each isolate to confirm its identity. Free-living and plant-parasitic nematodes from several field samples collected in California were extracted using the centrifugal flotation method and their extracts were used as background non-target DNA.

Nematode extracts
Several extracts were prepared from: (i) J2s, (ii) J2s with other non-target nematodes, (iii) females and (iv) plant gall tissue with one or more females and eggmasses. The J2 were placed in distilled water on a microscope slide. The nematodes were cut using a dental needle under a stereo microscope and put into 0.2 ml PCR tube with a total volume of 10 μ l. This stock crude extract was used to make a series of dilutions sequentially: 1:10, 1:20, 1:50, 1:100, 1:1,000 in water. Crude extract of several hundred non-target, soil free-living (cephalobids, diplogasterids and others) and plant-parasitic nematodes (hoplolaimids, pratylenchids and others) were also obtained by cutting the samples on a microscope slide using a dental needle.

RPA primer design and testing
Several forward and reverse RPA primers and probes were manually designed in this study. They included: (i) group-specific primers and probes for the tropical RKN complex, which were designed based on polymorphism of the IGS rRNA gene and (ii) species-specific primers and probes to M. javanica only, which were designed based on polymorphism of a genome fragment previously characterized by Hu et al. (2011). Primers were synthesized by Integrated DNA Technologies, Inc. (CA, USA). Eleven primer sets were screened in different combinations using the TwistAmp ® Basic kit (TwistDx, Cambridge, UK). Reactions were prepared according to the manufacturer's instructions. Detailed protocol is described by Subbotin and Burbridge (2021). The lyophilized reaction pellets were suspended in 29.5 μ l of rehydration buffer, 2.4 μ l of each forward and reverse primers (10 μ M) ( Table 2), 1 μ l of DNA template or nematode extract and 12.2 μ l of distilled water. For each sample, 2.5 μ l of 280 mM magnesium acetate was added to the lid of the tube and the lids were closed carefully. The tubes were inverted 10 to 15 times and briefly centrifuged to initiate reactions simultaneously. Tubes were incubated at 39°C (4 min) in MyBlock Mini Dry Bath (Benchmark Scientific, USA), inverted 10 to 15 times, briefly centrifuged and returned to the incubator block (39°C) for 20 min. Sample tubes were then placed in a freezer to stop the reaction. Amplification products were purified with the QIAquick PCR Purification Kit (Qiagen, USA). Five μ l of purified product were run in a 1% TAE buffered agarose gel (100 V, 60 min) and visualized with Gel Green stain (Biotium, USA). Amplification products were directly sequenced by Genewiz (CA, USA).

Real-time RPA assay
TwistAmp ® exo probes for the tropical RKN complex and M. javanica specific detection were designed according to the manufacturer's instructions and were synthesized by Biosearch Technologies, Inc (Petaluma, CA) ( Table 2). The real time detection of RPA assay products was accomplished using the TwistAmp ® exo kit (TwistDx, Cambridge, UK). Detailed protocol is described by Subbotin and Burbridge (2021). The reaction tubes were incubated at 39°C for 5 min, then inverted 10 to 15 times to mix, and briefly centrifuged. The tubes were immediately placed in Applied Biosystems™ QuantStudio™ 7 Flex Real-Time PCR System to incubate at 39°C for 15 min. The

LF-RPA assay
TwistAmp ® nfo probes for the tropical RKN complex and M. javanica specific detection were designed according to the manufacturer's instruction. LF-RPA assay products were accomplished using the TwistAmp ® nfo kit (TwistDx, Cambridge, UK). The reaction mixture for each RPA assay was prepared according to manufacturer's instructions. Detailed protocol is described by Subbotin and Burbridge (2021). The reaction tubes were incubated at 39°C for 4 min, then inverted 10 to 15 times to mix, briefly centrifuged and returned to the incubator block at 39°C for 16 min. Tubes were placed in freezer to stop reaction. For visual analysis with the Milenia® Genline Hybridetect-1 strips (Milenia Biotec GmbH, Germany), a testing solution containing 48 μ l of HybriDetect assay buffer and 12 μ l of the sample RPA product was prepared in a 0.5 ml PCR tube. Ten μ l of the testing solution was placed directly onto the sample area of the dipstick. Dipsticks was placed upright into 100 μ l of assay buffer and visual results were observed within 5 min. The amplification product was indicated by the development of a colored test line and/or a control line to confirm that the system worked properly. Two replicates of each variant were performed for sensitivity and specificity experiments.

Results
RPA diagnostic assay for the tropical RKN complex

RPA detection assay with visualization on gel
The group-specific forward Mtrop-RPA-F primer was designed and tested with the reverse Meloid-RPA-R primer proposed by Subbotin (2019). This primer set produced a band of ~ 313 bp in a length in RPA reactions using TwistAmp ® Basic kit with nematode extracts and DNA from species belonging to the tropical RKN complex. No band of this size was observed on gels with other species from nontropical RKN complex. The sequences of primers and probes used for the assays are listed in Table 2.

Real-time RPA detection assays
Based on the results of ten experimental runs, the threshold level for reliable tropical group detection was established as equal to 18 cycles (~6 min) with a baseline of 250,000 (∆Rn) fluorescence using the TwistAmp ® exo kit on the Applied Biosystems™QuantStudio™7 Flex Real-Time PCR System (Fig. 1). Samples that produced an exponential amplification curve above the threshold were considered as positive for the tropical RKN complex and below the threshold were considered as negative. Detections of the tropical RKN complex species were confirmed with all samples containing extracts and DNA from these species: M. arenaria, M. floridensis, M. hispanica, M. incognita, M. javanica and M. paranaensis (Fig. 1A). This assay did not detect M. haplanaria and M. enterelobii (data not shown). Sensitivity assay tests were run with serial dilutions of crude J2 extractions. The reliable detection level was estimated at 1/100 of a J2 per reaction tube (Fig. 1B). The results also showed a reliable detection of a single J2 with DNA extracted from 10 and 20 non-target nematodes and extracts from root galls containing females with eggs-masses belonging to the tropical RKN complex (data not shown).

LF-RPA detection assay
Lateral flow detection of RPA products using the TwistAmp®exo kit also showed specific and sensitive results. Positive test lines on the LF strips were observed for all the tropical RKN complex samples (M. arenaria, M. floridensis, M. hispanica, M. incognita, M. javanica and M. paranaensis), whereas samples with other nematode species showed only a control line ( Fig. 2A). The results of RPA assays showed reliable detection with a sensitivity of 1/100 of a J2 (Fig. 2B) and 1/10 of a female (data not shown) in reaction tubes. The results of RPA assay also showed a reliable detection of a single J2 with DNA extracted from10 and 20 non-target nematodes (data not shown) and extracts from root galls containing females with eggs-masses belonging to the tropical RKN complex (data not shown).

RPA detection assay with visualization on gel
The genome fragment of M. javanica with GenBank accession number JN005834 were aligned with similar genome fragments of this species (RCFK01023427; CEWN0101050) and other RKN species (RCFN010 05875-M. floridensis; RCFL01003500-M. incognita). Eleven primers were designed based on species polymorphism for M. javanica and then screened for the best performance in RPA with the TwistAmp®Basic kit. The species-specific forward Mj-RPA-F primer proposed by Ju et al. (2019) and the species-specific reverse RPA-mj-R1 primer were found to produce a species-specific band for M. javanica with a length of 213 bp and had no cross-reactions with other RKN. The sequences of primers and probes used for the assays are listed in Table 2.

Real-time RPA detection assays
Based on the results of twelve experimental runs, the threshold level for reliable M. javanica detection was established as approximately to 10 and up to 16 cycles (~3.5-5 min) with a baseline of 500,000 (∆Rn) fluorescence using the TwistAmp ® exo kit on the Applied Biosystems™QuantStudio™7 Flex Real-Time PCR System (Fig. 3). Samples that produced an exponential amplification curve above the threshold were considered as positive for M. javanica and below the threshold were considered as negative. Species-specific detection was confirmed with most samples containing extracts or DNA of this species, except for the two samples originated from California (Table 1; Fig. 3A) which gave negative results in some replicates. Additional testing revealed that this sample cannot be consistently identified as M. javanica using the present RPA assay.
The sensitivity assay tests were run with serial dilutions of crude J2 extractions. The reliable detection level of M. javanica was estimated at 1/100 of a J2 per a RPA reaction tube although the detection level was 1/1000 of a J2 in some replicates (Fig. 3B).
The detection of a single J2 for M. javanica was confirmed in the presence of background crude extracts from 10 and 20 non-target nematodes. No decrease in fluorescent signals was observed between these variants (data not shown).

LF-RPA assay
Lateral flow detection of RPA products also showed specific and sensitive results. Positive test lines on the LF strips were observed for most M. javanica samples, except for two samples from California. Samples with other nematode species showed only a control line (Fig. 4). The results of RPA assays showed reliable detection with a sensitivity of 1/20 of a J2 (Fig. 4). The detection of J2 for M. javanica was confirmed in the presence of background crude extract from 10 and 20 non-target nematodes (data not shown).

Discussion
The RKN control programs aim to reduce impact and distribution of these pests in agricultural fields and nurseries. However, effective control and eventual elimination of these nematodes needs high sensitive diagnostic tests. In this study we developed a rapid, reliable and affortable method of detection for RKN belonging to the tropical complex using RPA detection technology. The entire detection process for real-time RPA assay can be completed within approximately 16 min, including 4 min for crude nematode extract preparation, 11 min (5 + 6) for the RPA reaction with 1 min for mixing and centrifugation of tubes. The entire detection process for the LF-RPA assay can be completed within approximately 30 min, including  (B) Sensitivity assay with a crude J2 extract of M. javanica (isolate 40). Dilution series: 1/10 J2 per tube-4 and 11; 1/20 J2 per tube-1 and 9; 1/50 J2 per tube-5 and 8; 1/100 J2 per tube-3 and 7; 1/1000 J2 per tube-6 and 14; positive control-2 and 10; negative control-13. The vertical line on a graph: fluorescence ∆Rn. ∆Rn is calculated at each cycle as ∆Rn (cycle) = Rn (cycle)-Rn (baseline), where Rn = normalized reporter. The horizontal line on a graph: cycles, each cycle = 20 sec. 4 min for crude nematode extract preparation, 20 min (4 + 16) for the RPA reaction, 1 min for mixing and centrifugation of tubes, and 5 min for visual detection on the LF strips. This calculation does not include the time for preparation of the RPA reaction mixture.
In this study we developed the tropical RKN complex group-specific RPA assay, which allowed making preliminary screening to detect the presence of any species from this group in samples. It should be noticed that J2 of the RKN could be easily misidentified with Tylenchidae and other nematode juveniles and this assay can help to make a correct identification. To develop species-specific RKN RPA assay in the lab, several species-specific primers for M. incognita, M. arenaria and M. javanica were tested, but only species-specific primers of M. javanica showed species-specific amplification and the speciesspecific assay was only developed to this species in the frame of this project.
For desinging the species-specific M. javanica RPA primers and probe we used a genome fragment firstly proposed by Randig et al. (2002) and then applied for RPA assays by Ju et al. (2019) and Chi et al. (2020). In our RPA assay the forward speciesspecific RPA primer proposed by Ju et al. (2019) and a new reverse primer were used. This primer set was species-specific for M. javanica only. Although, the test specifically identified a wide range of M. javanica samples, it did not detect two populations of M. javanica from California. Further testing of more M. javanica populations as well as testing with low number of nematodes in samples is needed to further assess the performance of this assay.
Advantages and disadvantages of the RPA method for nematode diagnostics are discussed in our publications (Subbotin, 2019;Subbotin and Burbridge, 2021). Sensitivity of the present assays is comparable with those already developed by Ju et al. (2019) and Chi et al. (2020). However, our assays do not requre DNA extraction and allow for detection of nematodes in plant tissues and in a mixure with other nematodes. RPA assay has great potential for diagnosing the RKN in the lab, field or in areas with a minimal laboratory infrastructure.