Simultaneous detection and discrimination of echinococcus granulosus and ecchinococcus multilularis using real-time polymerase chain reaction and high-resolution melting analysis

Echinococcosis is a zoonotic disease caused by the tapeworm Echinococcus. The two most common species are E. granulosus and E. multilocularis. They cause infections in humans called cystic echinococcosis (CE) and alveolar echinococcosis (AE), respectively. Due to current epidemiological trends, there is a growing need for a sensitive and specific assay that can distinguish between the two infections. The purpose of this research was to design a multiplex PCR assay for serum that will be able to simultaneously identify and distinguish between E. granulosus and E. multilocularis via high-resolution melting analysis (HRMA). A primer set was designed to amplify the mitochondrial ND5 gene of E. multilocularis and a previously designed and tested primer set was used to amplify a genomic repeat in E. granulosus known as EgG1 Hae III. Human DNA was used as the positive internal control along with previously designed primers targeting the CFTR gene. All templates and primer sets were combined into a multiplex reaction. Optimization was achieved by varying the primer concentrations to achieve equal amplification of all targets. Serial dilution of all three templates was carried out. Each concentration of Echinococcus template was tested individually in combination with each concentration of control DNA to establish a limit of detection for each organism and an appropriate amount of control DNA to be used in the assay. iv To eliminate bias from the interpretation of results, 20 blind samples were tested. Each consisted of one of four concentrations of either Echinococcus template or water. Results were reported as E. multilocularis positive, E. granulosus positive or negative. The samples were de-blinded and compared to the results obtained. Eighteen out of 20 results were identified correctly. The two samples that were not identified correctly were called negative, but had very low concentrations of either E. granulosus or E. multilocularis template. Further research should be conducted to find a more suitable positive control due to its preferential amplification. However, this assay shows promise in its ability to detect very low levels of Echinococcus DNA and may have clinical use in the future.