Identification of Echinococcus granulosus Genotypes G1 and G3 by SNPs Genotyping Assays.

Piero Bonelli,Cinzia Santucciu,Silvia Dei Giudici,Giovanna Masala,Lorena Mura,Angela Peruzzu,Caterina Maestrale

doi:10.3390/pathogens10020125

Piero Bonelli, Cinzia Santucciu + Show 5 more

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https://doi.org/10.3390/pathogens10020125

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Abstract

Echinococcus granulosus sensu lato (s.l.) is the causative agent of cystic echinococcosis in animals and humans. Different E. granulosus s.l. genotypes exhibit great diversity in their life cycle, host selectivity and pathogenicity. For this reason, the study of genetic variation within Echinococcus species is of importance for their epidemiological implication. We employed two SNP genotyping technologies to distinguish G1 and G3 E. granulosus sensu stricto (s.s.). genotypes. The genotypes of DNA samples (n = 28) extracted from hydatid cysts of different animal species were identified by amplification and sequencing of a fragment of the mitochondrial nad5 gene. Two SYBR green and three TaqMan real time PCR assays were developed for targeting of three nad5 informative positions (SNP758, 1123, and 1380) known to be able to discriminate G1 from G3. Genotyping by SYBR Green PCR based on cycle threshold (Ct) with melting temperature (Tm) analysis and performed on SNP1123 and SNP1380 failed to identify one DNA sample. TaqMan assays for SNP758, 1123 and 1380 effectively confirmed genotype identification obtained by Sanger sequencing. Our results demonstrated that the combination of the three Taqman assays developed in this study represents a valuable and cost effective tool alternative to DNA sequencing for E. granulosus s.s. genotyping.

Highlights

A fragment of the mt nad5 gene was successfully amplified and sequenced from 28 hydatid cysts samples collected in this study
DNA isolates characterized by the presence of G at the three informative positions were considered G1; whereas those with C at position 758, and with A at position 1123 and 1380, were assigned to G3 genotype
Template Control), FAM (5(6)-carboxyfluorescein), VIC (2 -chloro-7 phenyl-1,4-dichloro-6-carboxy-fluorescein). In this this study study we we aimed aimed to to identify identify an an efficient efficient single nucleotide polymorphisms (SNPs)-based

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.Licensee MDPI, Basel, Switzerland.Attribution (CC BY) license (https://creativecommons.org/licenses/by/The larval form of Echinococcus granulosus sensu lato (s.l.) is the etiological agent of cystic echinococcosis (CE). CE is a worldwide-distributed disease whose strong zoonotic character is cause of great concern for human health. Considering its consistent clinical and economic burden, the World Health Organization (WHO) included CE in a list of seven neglected zoonotic diseases requiring priority intervention [1]. The life cycle of

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