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A novel RFLP-PCR method for the rapid diagnosis of Echinococcus multilocularis and different Echinococcus granulosus sensu lato species from tissue samples
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DOI：10.1051/parasite/2026017.png)
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Abstract
En 中文
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. L'& eacute;chinococcose alv & eacute;olaire et l'& eacute;chinococcose kystique sont des zoonoses qui affectent chaque ann & eacute;e des milliers de personnes en Europe. Cinq esp & egrave;ces d'Echinococcus sont end & eacute;miques en Europe, mais leur importance relative chez l'homme et l'animal varie. Afin d'identifier ces esp & egrave;ces d'Echinococcus & agrave; partir d'& eacute;chantillons tissulaires, une m & eacute;thode RFLP-PCR a & eacute;t & eacute; d & eacute;velopp & eacute;e et valid & eacute;e conform & eacute;ment aux normes fran & ccedil;aises de qualit & eacute; pour le diagnostic mol & eacute;culaire (NF U47-600-2). L'amplification par PCR d'un fragment d'environ 846 pb du g & egrave;ne de l'ARN ribosomique 12S est suivie d'une digestion unique par trois enzymes de restriction (AccI, DdeI et HinfI). La m & eacute;thode RFLP-PCR permet de distinguer Echinococcus multilocularis, E. granulosus sensu stricto, E. equinus, E. ortleppi et E. canadensis gr & acirc;ce & agrave; six profils sp & eacute;cifiques compos & eacute;s de deux ou trois bandes. Chaque profil est sp & eacute;cifique de l'esp & egrave;ce, & agrave; l'exception d'un profil sp & eacute;cifique suppl & eacute;mentaire associ & eacute; au g & eacute;notype G8 d'E. canadensis. Les esp & egrave;ces appartenant & agrave; d'autres genres de la famille des Taeniidae correspondent & agrave; deux autres types de profils. De plus, des analyses bioinformatiques ont pr & eacute;dit trois profils sp & eacute;cifiques suppl & eacute;mentaires pour les quatre autres esp & egrave;ces d'Echinococcus. La limite de d & eacute;tection de la m & eacute;thode, extraction d'ADN incluse, est estim & eacute;e & agrave; au moins 1 000 copies. La sp & eacute;cificit & eacute; et la sensibilit & eacute; totales ont & eacute;t & eacute; obtenues en testant un panel de 73 et 79 & eacute;chantillons d'ADN pr & eacute;alablement identifi & eacute;s par s & eacute;quen & ccedil;age. Le faible co & ucirc;t, la simplicit & eacute; et la rapidit & eacute; de mise en oe uvre de cette technique RFLP-PCR la rendent adapt & eacute;e aux & eacute;tudes & eacute;pid & eacute;miologiques de grande envergure, ainsi qu'au diagnostic cibl & eacute; d'& eacute;chantillons tissulaires, tels que des kystes ou des vers, sans n & eacute;cessiter de s & eacute;quen & ccedil;age.
Keywords:
Echinococcus
Molecular diagnostic
RFLP-PCR
Larval stage
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