Table 1
Oligonucleotide sequences of LAMP primers targeting the cm gene for Globodera ellingtonae, Globodera rostochiensis, and Globodera pallida.
| Species | Primer | Type | Sequence (5′–3′) | Length (bp) | Tm | Target region length (bp) |
|---|---|---|---|---|---|---|
| Globodera ellingtonae | Ge-F3 | Forward outer | CCAGCAGATTCTTTTTTACATCA | 23 | 57.08 | ∼315 |
| Ge-B3 | Backward outer | AGTCGGCTTACAACACAT | 18 | 55.37 | ||
| Ge-FIP | Forward inner | ATTGCGCAAAATGATTTCCTCCGAAT ACTTCCCAAAAGATTACCTAAC | 48 | |||
| Ge-BIP | Backward inner | TGTGCCTTCATGAAGAACATTGATCCGTTGTCGATTTCGTT | 41 | |||
| Ge-LP | Loop | TCAAATTTGTCGTTGGGATGGAAGG | 25 | |||
| Globodera rostochiensis | Gr-F3 | Forward outer | TCTTCATTGTCGGCATGG | 18 | 56.54 | ∼297 |
| Gr-B3 | Backward outer | ACCTTTTTTACCTGAATGACTT | 22 | 55.59 | ||
| Gr-FIP | Forward inner | CAACCTTTTCCCGCTCGAAATCGTTG GCCAAAGATGTGGT | 40 | |||
| Gr-BIP | Backward inner | GCGAAGAGTGCCGGGATAAGAGCGTCCATTTGGTCTTG | 38 | |||
| Gr-LP | Loop | CAACTACGGGGAGCCGTTTT | 20 | |||
| Globodera pallida | Gp-F3 | Forward outer | TTTCCAAAAGATTGCCTAGC | 20 | 55.74 | ∼285 |
| Gp-B3 | Backward outer | CGTTTAGTGACCGTACCT | 18 | 55.06 | ||
| Gp-FIP | Forward inner | CAATGTTCTTCATGAAGGCACAGTCACAACAAATGCAAGTCATCG | 45 | |||
| Gp-BIP | Backward inner | CCAAACGGAAACGGAATCCCTGATTTGGCTTACAACACAT | 40 | |||
| Gp-LP | Loop | GGACTTGCGCAAAATGATTTCCTC | 24 |
Figure 1
Optimization of LAMP assay for Globodera ellingtonae. (a) amplification of the cm gene using species-specific LAMP outer primers (F3 and B3), resolved on 1.5% agarose gel. Lane:100 bp DNA ladder; Lanes 2 and 3: Globodera ellingtonae (Ge); lanes 4 and 5: Globodera rostochiensis (Gr); and lanes 6 and 7: Globodera pallida (Gp). (b) qPCR amplification curves for the cm gene of Globodera ellingtonae (Ge); Globodera rostochiensis (Gr); and Globodera pallida (Gp). (c) Optimization of LAMP amplification temperature from 56 to 66℃, analyzed on 1.5% agarose gel. The LAMP assay conduced at 62℃ produced the brightest bend on the gel. (d) Colorimetric closed tube LAMP assay at the amplification temperature from 56 to 66℃ visualized under visible light (top) and UV illumination (bottom). Here, positive reactions appear green, whereas negative reactions and non-template control (NTC) remain orange in color due to staining with SYBR Green I dye.
Figure 2
Optimization of LAMP assay for Globodera ellingtonae. (a) Optimization of LAMP amplification time from 30 to 90 min, analyzed on 1.5% agarose gel. The LAMP assay conducted at 30 min produced the brightest bend on the gel. (b) Colorimetric closed tube LAMP assay at the amplification temperature from 30 to 90 min visualized under visible light (top) and UV illumination (bottom). (c) Optimized LAMP reaction compared with NTC under visible light (top) and UV illumination (bottom). Here, positive reactions appear green, whereas negative reactions and NTC remain orange in color due to staining with SYBR Green I dye.
Table 2
Optimized LAMP reaction parameters for Globodera pallida, Globodera rostochiensis, and Globodera ellingtonae
| Optimization | Optimized LAMP reaction for G. pallida | Optimized LAMP reaction for G. rostochiensis | Optimized LAMP reaction for G. ellingtonae | |
|---|---|---|---|---|
| dNTPs (mM L−1) | 0.2–1.8 | 2.1 | 2.1 | 2.1 |
| MgSO4 (mM L−1) | 2–10 | 1.2 | 1.4 | 1.4 |
| Bst DNA Polymerase (U µL−1) | 0.04–0.12 | 0.08 | 0.08 | 0.08 |
| Temperature (℃) | 54–66 | 60 | 62 | 62 |
Figure 3
Specificity and sensitivity of the close-tube LAMP assay for G. ellingtonae. (a) Sensitivity of the LAMP assay using serially tenfold diluted gDNA of G. ellingtonae starting from 1 ng to 10 fg, visualized by 1.5% agarose gel electrophoresis. Each reaction contained 1 µL of template DNA per assay. The LAMP assay consistently detected till 100 fg. (b) Colorimetric closed tube LAMP assay visualized under visible light (top) and UV illumination (bottom). Here, positive reactions appear green, whereas negative reactions remain orange in color due to staining with SYBR Green I dye (c) qPCR amplification of serially diluted G. ellingtonae gDNA. (d) Specificity of the LAMP assay using G. ellingtonae specific primers evaluated against gDNA from G. pallida (Gp), G. rostochiensis (Gr), G. ellingtonae (Ge), and NTC. Amplification was resolved in 1.5% agarose gel. (e) Colorimetric closed tube LAMP assay visualized under visible light (top) and UV illumination (bottom). (f) qPCR specificity assay performed using G. ellingtonae specific primers with gDNA from G. ellingtonae, G. pallida, and G. rostochiensis.