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

Sergei A. Subbotin

Sergei A. Subbotin

Plant Pest Diagnostic Center, California Department of Food and AgricultureSacramento, USA
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Subbotin, Sergei A.
, 
Juan E. Palomares-Rius

Juan E. Palomares-Rius

Instituto de Agricultura Sostenible (IAS), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Excelencia Internacional AgroalimentarioCórdoba, Spain
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Palomares-Rius, Juan E.
 und 
Pablo Castillo

Pablo Castillo

Instituto de Agricultura Sostenible (IAS), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Excelencia Internacional AgroalimentarioCórdoba, Spain
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Castillo, Pablo
  
05. Juli 2024
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Artikel-Kategorie: Research Paper
Online veröffentlicht: 05. Juli 2024
Eingereicht: 12. Feb. 2024
DOI: https://doi.org/10.2478/jofnem-2024-0023
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© 2024 Sergei A. Subbotin et al., published by Sciendo
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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_

Species Sample code Origin Plant-host RPA experiments
Meloidogyne artiellia CD3476 Italy, Bari Wheat +
M. artiellia CD3477 Spain, Córdoba, Montalbán Chickpea +
M. artiellia CD3962 Spain, Granada, Alomartes Chickpea +
M. artiellia CD4107 Spain, Granada, Guadix Wheat +
M. artiellia CD4018 Spain, Jaen, Jamilena Wheat +
M. arenaria CD2444 USA, Florida Prunus persica −
M. arenaria CD3093 USA, Florida P. persica −
M. baetica CD3382 Spain Olea europaea ssp. silvestris −
M. christiei CD1471 USA Florida Turkey oak −
M. floridensis CD3324 USA, California P. dulcis −
M. floridensis CD2452 USA, Florida P. persica −
M. enterolobii CD3984 USA, Florida Guava −
M. enterolobii CD3991 USA, Florida Guava −
M. hapla CD3384 Moldova, Tiraspol Sweet pepper −
M. hapla CD2461 USA, Florida Strawberry −
M. haplanaria CD3446 USA, California Sarracenia sp. −
M. hispanica CD2435 USA, Florida Urena lobata −
M. hispanica CD3416 USA, Florida Caladium × hortulanum −
M. incognita CD3101 USA, California Solanum lycopersicum −
M. javanica CD3050 USA, Florida, P. persica −
M. javanica CD2355 USA, Florida Humulus lupulus −
M. naasi CD3381 Germany Grasses −
M. paranaensis CD3510 USA Caladium sp. −
Meloidogyne sp. CD3732 USA, Florida Unknown plant −

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

Primer or probe Sequence (5′ – 3′)
M-artiel-RPA-ITS-F1 T GGG GAC TGT CGA TTG TCG GTG CGT TCG
M-artiel-RPA-ITS-R1 AGC 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-exo A 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]
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