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Recombinase Polymerase Amplification assay for detection of the British root-knot nematode, Meloidogyne artiellia

Journal of Nematology
Volume 56 (2024): Issue 1 (March 2024)
By:
Open Access
|Jul 2024

Figures & Tables

Figure 1.

Chickpea roots with galls induced by Meloidogyne artiellia. Arrows indicate females on roots.
Chickpea roots with galls induced by Meloidogyne artiellia. Arrows indicate females on roots.

Figure 2.

The fragment of alignment of the ITS rRNA gene sequences for several root-knot nematodes with the positions of RPA primers and probes for M. artiellia detection used in this study.
The fragment of alignment of the ITS rRNA gene sequences for several root-knot nematodes with the positions of RPA primers and probes for M. artiellia detection used in this study.

Figure 3.

RPA assays using real-time fluorescent detection with examples of amplification plots. (A) Specificity assay with DNA samples of Meloidogyne spp. and crude second-stage juvenile (J2) extract of M. artiellia. Plot - 1: M. artiellia (CD4108, extract); 2: M. artiellia (CD3477); 3: M. hapla (CD3384); 4: M. hispanica (CD2435); 5: M. arenaria (CD3093); 6: M. floridensis (CD3324); 7: M. floridensis (CD2452); 8: M. arenaria (CD2444); 9: M. enterolobii (CD3991); 10: M. baetica (CD3382); 11: M. javanica (CD2355); 12: M. christiei (CD1471); 13: M. paranaensis (CD3510); 14: M. hapla (CD2461); 15, 16: negative control (no DNA). (B) Sensitivity assay with a dilution series of crude J2 extracts of M. artiellia. Plot - 1, 1’: J2 specimen per reaction tube; 2, 2’: 0.5 J2 specimen per reaction tube; 3, 3’: 0.25 J2 specimen per reaction tube; 4, 4’: 0.125 J2 specimen per reaction tube; 5, 6 - M. artiellia (CD3962), 1/20 female (dried) per reaction tube; 7–8: negative control (no DNA). The vertical line on a graph: fluorescence ∆Rn. ∆Rn is calculated at each cycle as DRn (cycle) = Rn (cycle) - Rn (baseline), where Rn = normalized reporter. The horizontal line on a graph: cycles, each cycle = 20 s.
RPA assays using real-time fluorescent detection with examples of amplification plots. (A) Specificity assay with DNA samples of Meloidogyne spp. and crude second-stage juvenile (J2) extract of M. artiellia. Plot - 1: M. artiellia (CD4108, extract); 2: M. artiellia (CD3477); 3: M. hapla (CD3384); 4: M. hispanica (CD2435); 5: M. arenaria (CD3093); 6: M. floridensis (CD3324); 7: M. floridensis (CD2452); 8: M. arenaria (CD2444); 9: M. enterolobii (CD3991); 10: M. baetica (CD3382); 11: M. javanica (CD2355); 12: M. christiei (CD1471); 13: M. paranaensis (CD3510); 14: M. hapla (CD2461); 15, 16: negative control (no DNA). (B) Sensitivity assay with a dilution series of crude J2 extracts of M. artiellia. Plot - 1, 1’: J2 specimen per reaction tube; 2, 2’: 0.5 J2 specimen per reaction tube; 3, 3’: 0.25 J2 specimen per reaction tube; 4, 4’: 0.125 J2 specimen per reaction tube; 5, 6 - M. artiellia (CD3962), 1/20 female (dried) per reaction tube; 7–8: negative control (no DNA). The vertical line on a graph: fluorescence ∆Rn. ∆Rn is calculated at each cycle as DRn (cycle) = Rn (cycle) - Rn (baseline), where Rn = normalized reporter. The horizontal line on a graph: cycles, each cycle = 20 s.

Figure 4.

Lateral flow recombinase polymerase amplification (LF-RPA) assay with examples of lateral flow strips. (A) Specificity assay with DNA samples of different nematodes. Strips: 1 - M. hispanica (CD2435); 2 - M. hispanica (CD3416); 3 - M. baetica (CD3382); 4 - M. javanica (CD2355); 5 - M. christiei (CD1471); 6 - M. hapla (CD2461); 7 - M. paranaensis (CD3510); 8 - Meloidogyne sp. (CD3732); 9 - M. enterolobii (CD3984); 10 - M. artiellia (CD3962). (B) Sensitivity assay with crude M. artiellia extract (CD3962). Strips: 1: M. artiellia (DNA control, CD3962); 2 – 1 J2 specimen per reaction tube; 3: 0.5 of J2 specimen per reaction tube; 4: 0.25 of J2 specimen per reaction tube; 5: 0.125 of J2 specimen per reaction tube; 6: 0.06 of J2 specimen per reaction tube; 7: 0.03 of J2 specimen per reaction tube; 8: negative control (no DNA).
Lateral flow recombinase polymerase amplification (LF-RPA) assay with examples of lateral flow strips. (A) Specificity assay with DNA samples of different nematodes. Strips: 1 - M. hispanica (CD2435); 2 - M. hispanica (CD3416); 3 - M. baetica (CD3382); 4 - M. javanica (CD2355); 5 - M. christiei (CD1471); 6 - M. hapla (CD2461); 7 - M. paranaensis (CD3510); 8 - Meloidogyne sp. (CD3732); 9 - M. enterolobii (CD3984); 10 - M. artiellia (CD3962). (B) Sensitivity assay with crude M. artiellia extract (CD3962). Strips: 1: M. artiellia (DNA control, CD3962); 2 – 1 J2 specimen per reaction tube; 3: 0.5 of J2 specimen per reaction tube; 4: 0.25 of J2 specimen per reaction tube; 5: 0.125 of J2 specimen per reaction tube; 6: 0.06 of J2 specimen per reaction tube; 7: 0.03 of J2 specimen per reaction tube; 8: negative control (no DNA).

Figure 5.

Lateral flow recombinase polymerase amplification (LF-RPA) assay with examples of lateral flow strips. Strips: 1 - M. artiellia (CD4108), 1 J2 per reaction tube; 2, 3 - M. artiellia (CD4107), 1/20 female (ethanol fixed) per reaction tube; 4 - M. artiellia (CD4107), 1 J2 per reaction tube, 5, 8 - M. artiellia (CD3962) dried females with plant root tissues; 6 - M. artiellia (CD3962), 1 J2 per reaction tube; 7 - M. artiellia (CD3962), 1/20 of female (dried) per reaction tube; 9: negative control (no DNA).
Lateral flow recombinase polymerase amplification (LF-RPA) assay with examples of lateral flow strips. Strips: 1 - M. artiellia (CD4108), 1 J2 per reaction tube; 2, 3 - M. artiellia (CD4107), 1/20 female (ethanol fixed) per reaction tube; 4 - M. artiellia (CD4107), 1 J2 per reaction tube, 5, 8 - M. artiellia (CD3962) dried females with plant root tissues; 6 - M. artiellia (CD3962), 1 J2 per reaction tube; 7 - M. artiellia (CD3962), 1/20 of female (dried) per reaction tube; 9: negative control (no DNA).

Samples of cyst-forming nematodes tested in the present study_

SpeciesSample codeOriginPlant-hostRPA experiments
Meloidogyne artielliaCD3476Italy, BariWheat+
M. artielliaCD3477Spain, Córdoba, MontalbánChickpea+
M. artielliaCD3962Spain, Granada, AlomartesChickpea+
M. artielliaCD4107Spain, Granada, GuadixWheat+
M. artielliaCD4018Spain, Jaen, JamilenaWheat+
M. arenariaCD2444USA, FloridaPrunus persica−
M. arenariaCD3093USA, FloridaP. persica−
M. baeticaCD3382SpainOlea europaea ssp. silvestris−
M. christieiCD1471USA FloridaTurkey oak−
M. floridensisCD3324USA, CaliforniaP. dulcis−
M. floridensisCD2452USA, FloridaP. persica−
M. enterolobiiCD3984USA, FloridaGuava−
M. enterolobiiCD3991USA, FloridaGuava−
M. haplaCD3384Moldova, TiraspolSweet pepper−
M. haplaCD2461USA, FloridaStrawberry−
M. haplanariaCD3446USA, CaliforniaSarracenia sp.−
M. hispanicaCD2435USA, FloridaUrena lobata−
M. hispanicaCD3416USA, FloridaCaladium × hortulanum−
M. incognitaCD3101USA, CaliforniaSolanum lycopersicum−
M. javanicaCD3050USA, Florida,P. persica−
M. javanicaCD2355USA, FloridaHumulus lupulus−
M. naasiCD3381GermanyGrasses−
M. paranaensisCD3510USACaladium sp.−
Meloidogyne sp.CD3732USA, FloridaUnknown plant−

RPA primers and probes used for amplification of DNA of Meloidogyne artiellia_

Primer or probeSequence (5′ – 3′)
M-artiel-RPA-ITS-F1T GGG GAC TGT CGA TTG TCG GTG CGT TCG
M-artiel-RPA-ITS-R1AGC GCC AGC CGT TCC ACA GCG GAA AGA
M-artiel-RPA-ITS-R1-biotin[Biotin] AGC GCC AGC CGT TCC ACA GCG GAA AGA
Probe-Mart-ITS1-nfo1[FAM]* CTT TAC CAT GTG ATG GTT ACA TTT CGA AAG TGC [THF] AAA ACA CAC TAT TAT T [C3-spacer]
Probe-Mart-ITS1-exoA TGG TTA CAT TTC GAA AGT GCC AAA ACA CAC [FAM-dT]AT [THF] AT [BHQ1-dT] TAT TAA GTG TTG TGT T [C3-spacer]
DOI: https://doi.org/10.2478/jofnem-2024-0023 | Journal eISSN: 2640-396X | Journal ISSN: 0022-300X
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Language: English
Submitted on: Feb 12, 2024
Published on: Jul 5, 2024
Published by: Society of Nematologists, Inc.
In partnership with: Paradigm Publishing Services
Publication frequency: 1 times per year
Keywords:
chickpea,
diagnostics,
ITS rRNA,
RPA,
species-specific primer
Related subjects:
Life sciences,
Life sciences, other

© 2024 Sergei A. Subbotin, Juan E. Palomares-Rius, Pablo Castillo, published by Society of Nematologists, Inc.
This work is licensed under the Creative Commons Attribution 4.0 License.

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