A novel RFLP-PCR method for the rapid diagnosis of Echinococcus multilocularis and different Echinococcus granulosus...

Both alveolar echinococcosis and cystic echinococcosis are zoonotic diseases affecting thousands of humans in Europe annually. Five Echinococcus species are known to be endemic in Europe, but with varying relative importance in humans and animals. In order to identify these Echinococcus species from tissue samples, an RFLP-PCR method was developed and validated in accordance with the French quality standards for molecular diagnostics (NF U47-600-2). Amplification by PCR of about 846 bp from the 12S ribosomal RNA gene was followed by a single digestion step with three restriction enzymes (AccI, DdeI and HinfI). The RFLP-PCR method distinguishes Echinococcus multilocularis, E. granulosus sensu stricto, E. equinus, E. ortleppi and E. canadensis in six specific profiles composed of two or three bands. Each profile is specific for the species, except an additional specific profile from genotype G8 of E. canadensis. Species from other genera of the Taeniidae family correspond to two other types of profiles. Additionally, in silico analyses predicted three specific additional profiles for the other four Echinococcus species. The limit of detection of the method including DNA extraction is estimated to be a minimum of 1,000 copies. Total specificity and sensitivity were obtained by testing a panel of 73 and 79 DNA samples previously identified by sequencing. This RFLP-PCR technique is cost-effective, simple and rapid to implement, making it suitable for use in large epidemiological studies, as well as for single diagnostic purposes targeting tissue samples, such as cysts or worms, without requiring sequencing.