LAMP Assay for the Detection of Echinococcus multilocularis Eggs Isolated from Canine Faeces by a Cost-Effective NaOH-Based DNA Extraction Method

Abstract

The detection of Echinococcus multilocularis in infected canids and the environment is pivotal for a better understanding of the epidemiology of alveolar echinococcosis in endemic areas. Necropsy/sedimentation and counting technique remain the gold standard for the detection of canid infection. PCR-based detection methods have shown high sensitivity and specificity, but they have been hardly used in large scale prevalence studies. Loop-mediated isothermal amplification (LAMP) is a fast and simple method to detect DNA with a high sensitivity and specificity, having the potential for field-application. A specific LAMP assay for the detection of E. multilocularis was developed targeting the mitochondrial nad1 gene. A crucial step for amplification-based detection methods is DNA extraction, usually achieved utilising silica-gel membrane spin columns from commercial kits which are expensive. We propose two cost-effective and straightforward methods for DNA extraction, using NaOH (method 1A) and InstaGeneTM Matrix (method 1B), from isolated eggs circumventing the need for commercial kits. The sensitivity of both assays with fox samples was similar (72.7%) with multiplex-PCR using protocol 1A and LAMP using protocol 1B. Sensitivity increased up to 100% when testing faeces from 12 foxes infected with more than 100 intestinal stages of E. multilocularis. For dogs, sensitivity was similar (95.4%) for LAMP and multiplex-PCR using protocol 1B and for both methods when DNA was extracted using protocol 1A (90.9%). The DNA extraction methods used here are fast, cheap, and do not require a DNA purification step, making them suitable for field studies in low-income countries for the prevalence study of E. multilocularis.

Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?

“…The PCR technique demonstrates high specificity in detecting the parasite's genetic material, but this is rarely used due to its complexity and cost. Consequently, researchers described an assay based on the LAMP method that detects the mitochondrial gene nad1 [56]. The sensitivity of this assay was very similar to PCR, but LAMP omitted the step of extracting the genetic material.…”

“…The sensitivity of this assay was very similar to PCR, but LAMP omitted the step of extracting the genetic material. Furthermore, LAMP had other advantages over PCR, especially in terms of analysis time, complexity, and field applicability [56].…”

Development of a New LAMP Assay for the Detection of Ancylostoma caninum DNA (Copro-LAMPAc) in Dog Fecal Samples

“…The LAMP assay for E. granulosus s. s. DNA detection developed by Ni et al ( 70 ), was able to detect 10 pg of gDNA, and was more sensitive than both copro-ELISA and copro-PCR. While Bucher et al ( 71 ) developed an easy and cost-effective protocol for E. multilocularis copro-detection, some LAMP reactions for the detection of nematode DNA were not able to overcome the sensitivity values of gold standard techniques ( 72 ) or PCR methods ( 73 ).…”

First isolation of Echinococcus granulosus sensu lato genotype 7 in the archipelago of Cape Verde

“…All samples were centrifuged at 200 g for 10 min and kept in 1.5 mL tubes at −20°C. Using an alkaline lysis method, as described in Bucher et al (2021), genomic DNA was extracted from all the microscopically taeniid egg-positive samples and randomly selected 40% of the negative samples from each district, to document the sensitivity of the microscopic evaluation. Briefly, eggs were treated with 0.2 M NaOH, incubated at +95°C for 10 min and then 1 part of the supernatant was diluted in 50 parts of 100 mM Tris-HCl which was used as a template for the polymerase chain reaction (PCR).…”