Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Globodera ellingtonae

Chandni Shah; Louise-Marie Dandurand

doi:10.2478/jofnem-2026-0017

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Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Globodera ellingtonae

Journal of Nematology

Volume 58 (2026): Issue 1 (February 2026)

By: Chandni Shah and Louise-Marie Dandurand

Open Access

|Jul 2026

Figures & Tables

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.

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.

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.

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

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

Articles in this issue

DOI: https://doi.org/10.2478/jofnem-2026-0017 | Journal eISSN: 2640-396X | Journal ISSN: 0022-300X

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Language: English

Page range: 208 - 219

Submitted on: Mar 30, 2026

Accepted on: May 23, 2026

Published on: Jul 7, 2026

Published by: Society of Nematologists, Inc.

In partnership with: Paradigm Publishing Services

Publication frequency: 1 issue per year

Keywords:

Chorismate mutase gene,

Globodera ellingtonae ,

Loop-mediated isothermal amplification,

Potato cyst nematode

Related subjects:

Life sciences,

Life sciences, other

© 2026 Chandni Shah, Louise-Marie Dandurand, published by Society of Nematologists, Inc.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Volume 58 (2026): Issue 1 (February 2026)

Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Globodera ellingtonae

Figures & Tables

Figure 1

Figure 2

Figure 3

Optimized LAMP reaction parameters for Globodera pallida, Globodera rostochiensis, and Globodera ellingtonae

Oligonucleotide sequences of LAMP primers targeting the cm gene for Globodera ellingtonae, Globodera rostochiensis, and Globodera pallida_

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