Aug 29, 2025

Public workspaceStandardization of the technique 'LOOP-MEDIATED ISOTHERMAL AMPLIFICATION' (LAMP) using the target 18S rDNA for cutaneous leishmaniasis V.2

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvw4dj9lmk/v2
  • Elizabeth Cristina Araujo Ferreira Faulhaber1,
  • Daniel Moreira Avelar2,
  • Gabrielle Baldan Stanciola1,
  • Luanna da Silva Ventura1,
  • Andreza Pain Marcelino1,
  • maria.pimentel 1
  • 1Instituto Nacional de Infectologia Evandro Chagas - INI - FIOCRUZ RJ;
  • 2Instituto René Rachou - FIOCRUZ Minas
Elizabeth Cristina Araujo Ferreira Faulhaber
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvw4dj9lmk/v2
Protocol Citation: Elizabeth Cristina Araujo Ferreira Faulhaber, Daniel Moreira Avelar, Gabrielle Baldan Stanciola, Luanna da Silva Ventura, Andreza Pain Marcelino, maria.pimentel 2025. Standardization of the technique 'LOOP-MEDIATED ISOTHERMAL AMPLIFICATION' (LAMP) using the target 18S rDNA for cutaneous leishmaniasis. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw4dj9lmk/v2Version created by Elizabeth Cristina Araujo Ferreira Faulhaber
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 28, 2025
Last Modified: August 29, 2025
Protocol Integer ID: 225696
Keywords: 18S rDNA, “Loop-mediated isothermal amplification” , LAMP, Molecular Biology , Cutaneous Leishmaniasis, mediated isothermal amplification, leishmania sample, cutaneous leishmaniasis leishmaniasi, cutaneous leishmaniasi, isothermal amplification, several protocols for molecular testing, reactivity for all leishmania sample, molecular testing, genus leishmania, leishmaniasi, amplifying large amount, polymerase enzyme, protozoa, large amounts of dna, performance of the lamp technique, promising alternative for the diagnosis, amplification, lamp technique
Funders Acknowledgements:
National Institute of Infectology Evandro Chagas
Grant ID: INI - FIOCRUZ
René Rachou Institute -FIOCRUZ
Grant ID: FIOCRUZ MG
Coordination for the Improvement of Higher Education Personnel
Grant ID: CAPES
Abstract
Leishmaniasis are infectious diseases, transmitted by vectors, caused by protozoa of the genus Leishmania. Several protocols for molecular testing (conventional PCR - cPCR; and real-time PCR - qPCR) have been evaluated for the diagnosis of cutaneous leishmaniasis (CL), but they are limited to reference centers. A breakthrough in the diagnosis of various infectious diseases has been the development of the technique 'loop-mediated isothermal amplification' - LAMP. This method is capable of amplifying large amounts of DNA under isothermal conditions within 30-60 minutes. Our goal was to evaluate the performance of the LAMP technique targeting 18S rDNA using the WarmStart‱ Colorimetric LAMP 2x Master Mix (WS) kit and the Bst 2.0 DNA Polymerase enzyme with SYBR‱ Green I (BstSg). The analytical sensitivity (detection limit) with the WS mix was up to 10 fg in agarose gel and 100 fg visually, with consistent results in duplicates; and using BstSg, the sensitivity was up to 10 pg. In the analytical specificity using WS, there was reactivity for all Leishmania samples. However, the samples of Paracoccidioides brasiliensis (Pb) and Sporothrix globosa (Sg) showed bands in one of the duplicates in the gel, suggesting possible non-specific amplification. Using BstSg, visual reactivity was observed only for L. braziliensis (Lb). However, in agarose gel, samples of L. infantum (Li), L. guyanensis (Lg), and one of the duplicates of L. amazonensis (La) showed bands suggestive of amplification. The LAMP is a promising alternative for the diagnosis of leishmaniasis.
Image Attribution
LaPClinVigiLeish - INI - FIOCRUZ

Materials
EQUIPAMENTS
  • Thermo Scientific‱ NanoDrop‱ 2000 spectrophotometer
  • Double boiler water Novus N1030T - New Technique
  • 100 µL micropipette
  • 10 µL micropipette

MATERIALS
  • Sterile microtubes of 1.5 to 2 mL
  • Sterile microtubes of 0.5 mL

REAGENTS
  • Phosphorylated Deoxyribonucleotides dNTP's
  • Magnesium
  • Buffer 1X
  • Primers
  • Betaine
  • Bacillus stearothermophilus (BST)
  • SYBR‱ Green I
  • kit WarmStart‱Colorimetric LAMP 2x Master Mix
  • Water
  • Deoxyribonucleic acid (DNA)



Troubleshooting
Standardization assay
Standardization assay of LAMP with DNA from reference samples and negative controls using the commercial Warmstart kit (WS) targeting 18S rDNA, with an incubation time of 40 minutes. Tubes containing LAMP reactions with WS for the target 18S rDNA. Lb: Leishmania (Viannia) braziliensis; Li: leishmania (Leishmania) infantum; La: Leishmania (Leishmania) amazonensis. CN: negative control with DNA from a negative leishmaniasis patient. B: Negative control with water. (+) Positive reaction; (-) Negative reaction.


Standardization assay of LAMP with DNA from reference samples using WS lot 10265054 targeting 18S rDNA, with incubation times of 25, 30, 35, and 40 minutes. Tubes containing LAMP reactions with WS for the target 18S rDNA. Lb: Leishmania (Viannia) braziliensis; CN: negative control with DNA from a patient negative for leishmaniasis. B: Negative control with water. (+) Positive reaction; (-) Negative reaction.


Analytical Sensitivity
Analytical sensitivity with WS mix for target 18S rRNA, using a reference sample of Leishmania (Viannia) braziliensis in serial dilutions starting from 1 nanogram. Fig.A.Tubes containing LAMP reactions with WS for the target 18S rDNA at concentrations of 1ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg. CN: control containing DNA from a patient with a final diagnosis different from leishmaniasis. FigB: 2% agarose gel of the products obtained in the tubes illustrated in the reaction of FigA. PM: Molecular weight.


A
Analytical sensitivity test with Bst 2.0 DNA Polymerase com SYBR‱ Green I (BstSg) for the target 18S rRNA, using a reference sample of Leishmania (Viannia) braziliensis in serial dilutions starting from 1 nanogram. FigA:Tubes containing LAMP reactions with BstSg for the target 18S rDNA at concentrations of 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg. CN: control containing DNA from a patient with a final diagnosis different from leishmaniasis; B-Blank: Negative control with water. FigB: Electrophoresis on polyacrylamide gel of the products obtained from the tubes illustrated in the reaction of FigA. PM: Molecular weight.


Analytical specificity
Analytical specificity for the target 18Tubes containing LAMP reactions with WS for the target 18S rDNA at a concentration of 5ng/µL. FigA: Paracoccidioides brasiliensis (Pb), Sporothrix schenckii (Ss), Sporothrix braziliensis (Sb), Sporothrix globosa (Sg), Leishmania braziliensis (Lb), Leishmania (Viannia) lainsoni (Ll). CN: control containing DNA from a patient with a final diagnosis different from leishmaniasis. B - Blank: Negative control with water. FigB: 2% agarose gel of the products obtained in the tubes illustrated in the reaction of FigA. PM: Molecular weight.


Analytical specificity for the target 18S rDNA using WS and reference samples of Leishmania spp. Tubes containing LAMP reactions with WS for the target 18S rDNA at a concentration of 5ng/µL. FigA.:Leishmania infantum (Li), Leishmania (Viannia) guyanensis (Lg), Leishmania amazonensis (La), and Leishmania (Viannia) shawi (Ls). CN: control containing DNA from a patient with a final diagnosis different from leishmaniasis. B - Blank: Negative control with water. FigB.: 2% agarose gel of the products obtained in the tubes illustrated in the reaction of Fig.A. PM: Molecular weight.


Analytical specificity for the target 18S rRNA using BstSg and reference samples of Paracoccidioides spp., Sporothrix spp. and Leishmania spp. FigA.:Tubes containing LAMP reactions with BstSg for the target 18S rDNA at a concentration of 5ng/µL. Paracoccidioides brasiliensis (Pb), Sporothrix schenckii (Ss), Sporothrix brasiliensis (Sb), Sporothrix globosa (Sg), Leishmania braziliensis (Lb). CN: control containing DNA from a patient with a final diagnosis different from leishmaniasis. B - White: Negative control with water. Fig.B: 2% agarose gel of the products obtained in the tubes illustrated in the reaction of FigA. PM: Molecular weight.


Analytical specificity for the target 18S rRNA using BstSg, samples of Leishmania spp. FigA.: Tubes containing LAMP reactions with BstSg for the target 18S rDNA at a concentration of 5ng/µL. Leishmania infantum (Li), Leishmania guianensis (Lg), Leishmania amazonensis (La), and Leishmania shawi (Ls). CN: control containing DNA from a patient with a final diagnosis different from leishmaniasis. B-Blank: Negative control with water. Fig.B: 2% agarose gel of the products obtained from the tubes illustrated in the reaction of Fig.A. PM: Molecular weight.


Performance analysis
Performance analysis of BstSg using 18S rDNA in samples of DNA from swabs of skin lesions extracted with a commercial kit and by boiling without dilution and with dilution at 1-10, 1-20 1-50 and 1-100. SD: without dilution; CN: control containing DNA from a patient with a final diagnosis different from leishmaniasis; B-Branco: Negative control with water; CP: Positive control of L.braziliensis; (+) Positive reaction; (-) Negative reaction. Marked in blue: DNA samples extracted with the Pure Link‱ Genomic DNA Mini Kit (30ng/µL) and marked in red: DNA samples extracted by boiling (223,9 ng/µL).


Protocol references
ADAMS ER et al. 2010. Development of a Reverse Transcriptase Loop-Mediated Isothermal Amplification (LAMP) Assay for the Sensitive Detection of Leishmania Parasites in Clinical Samples. Am. J. Trop. Med. Hyg.82(4), pp. 591–596.

ADAMS ER et al. 2018. Development and evaluation of a novel loop-mediated isothermal amplification assay for diagnosis of cutaneous and visceral leishmaniasis. J Clin Microbiol.

CELESTE, J. L. L. et al. 2019.Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Leishmania amazonensis in skin samples. Exper. Parasitol., v. 203, p. 23–29.

DE FARIA VCS, et al. 2022. Impact assessment of different DNA extraction methods for non- invasive molecular diagnosis of tegumentary leishmaniasis. Acta Trop.

ERBER AC, Sandler PJ, de Avelar DM, Swoboda I, Cota G, Walochnik J. 2022. Diagnosis of visceral and cutaneous leishmaniasis using loop-mediated isothermal amplification (LAMP) protocols: a systematic review and meta-analysis. Parasit Vectors. Jan 24;15(1):34. doi: 10.1186/s13071-021-05133-2. PMID: 35073980; PMCID: PMC8785018.

IBARRA-MENESES, A. V. et al. 2021. Loop-mediated isothermal amplification allowsrapid, simple and accurate molecular diagnosis of human cutaneous and visceral leishmaniasis caused by Leishmania infantum when compared to PCR. Microorganisms, v. 9, n. 3, p. 1–7.

NOTOMI, T. et al. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Ac. Res., v. 28, n. 12, p. 63e–663.

NZELU, C. et al. 2014. Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection. Acta tropica, v. 132, n. 1, p. 1–6.

NZELU C et al. 2016. A rapid molecular diagnosis of cutaneous leishmaniasis by colorimetric malachite green-loop-mediated isothermal amplification (LAMP)combined with an FTA card as a direct sampling tool. Acta Tropica. v. 153, p.116–119.