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 InstaGene^TM 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.

Keywords: DNA extraction; Echinococcus; LAMP; NaOH; PCR; taeniid egg isolation.

Figure 1

Schematic representation of the process of field samples analysed in the present study. Step 7 shows the two methods used for DNA extraction in this study (protocols 1A and 1B). The visualisation of multiplex-PCR occurred after electrophoresis and in the case of LAMP results were assessed after change of colour of the reaction, electrophoresis, and lateral flow dipsticks as shown in step 9.