Development and evaluation of real-time loop-mediated isothermal amplification assay for rapid detection of cystic echinococcosis.

Mohamed E Ahmed,Fatima M Elamin,Martin P Grobusch,Ibtisam A Ali,Mawahib H Eldigail,Imadeldin E Aradaib

doi:10.1186/s12917-016-0809-2

Abstract

BackgroundCystic echinococcosis (CE) or hydatidosis, caused by the larval stage of Echinococcus granulosus (EG)-complex, is a neglected parasitic disease of public health importance. The disease is endemic in many African and Mediterranean countries including the Sudan. The objective of the present study was to develop and evaluate a real-time loop-mediated isothermal amplification (LAMP) assay for simple and rapid detection of CE in humans and domestic live stock in Sudan.MethodsA set of six LAMP primers, designed from the mitochondrial NADH-1 gene of EG cattle strain of genotype 5 (G5), was used as a target for LAMP assay. The assay was performed at a constant temperature (63 °C), with a real-time follow-up using a LightCycler and fluorochrome dye. Following amplification cycles in a simple water bath, LAMP products were observed for color change by naked eye and were visualized under UV light source using agarose gel electrophoresis.ResultsThe real-time LAMP assay identified a variety of hydatid cysts strains recovered in the Sudan, including Echinococcus canadenses (G6) and Echinococcus ortleppi (G5). Real-time LAMP positive results were detected by the presence of an amplification curve, whereas negative results were indicated by absence of fluorescence detection. Positive LAMP results appeared as a bluish-colored reaction as observed by naked eye, whereas negative LAMP results were observed as purple-colored reaction. The sensitivity studies indicated that the LAMP assay detected as little as a 10 fg of parasite DNA. There was 100 % agreement between results of the LAMP assay and our previously described nested PCR when testing 10-fold serial dilution of DNA extracted from EG-complex hydatid cyst. However, there was no cross-reactivity with other parasites including cysticercus bovis, Fasciola gigantica, and Schistosoma bovis and nucleic acid free samples.ConclusionThe developed LAMP assay would be expected to prove highly significant in epidemiological surveys of CE in developing countries or areas of resource-poor settings for both ease of use and cost.

Highlights

Cystic echinococcosis (CE) or hydatidosis, caused by the larval stage of Echinococcus granulosus (EG)complex, is a neglected parasitic disease of public health importance
Optimization condition and visualization of loop-mediated isothermal amplification (LAMP) product The optimization condition and visualization of LAMP products were determined using 10 pg of DNA extracted from Sudanese cattle strain (G5), which was incubated at a range of 60 to 65 °C
Positive LAMP result was indicated by the presence of amplification curve where as negative result was indicated by absence of fluorescence detection

Summary

Introduction

Cystic echinococcosis (CE) or hydatidosis, caused by the larval stage of Echinococcus granulosus (EG)complex, is a neglected parasitic disease of public health importance. Three EG-complex genotypes including, the sheep (G1), the cattle (G5) and the camel (G6) strains were reported in humans and livestock in the Sudan [8,9,10]. In the past few years CE has been repeatedly reported as an important emerging infectious parasitic disease in Central Sudan [1, 27, 28] It is, becoming increasingly obvious that the development of a simple and rapid molecular assay for detection of EG-complex is urgently needed in remote areas with resource-poor settings

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