Bonelli Piero, Dei Giudici Silvia, Peruzzu Angela, Mura Lorena, Santucciu Cinzia, Maestrale Caterina, Masala Giovanna
OIE Reference Laboratory for Echinococcosis, National Reference Center for Echinococcosis (CeNRE), IZS della Sardegna, 07100 Sassari, Italy.
Laboratory of Diagnostic Virology, Istituto Zooprofilattico della Sardegna (IZS), 07100 Sassari, Italy.
Pathogens. 2021 Jan 26;10(2):125. doi: 10.3390/pathogens10020125.
() is the causative agent of cystic echinococcosis in animals and humans. Different genotypes exhibit great diversity in their life cycle, host selectivity and pathogenicity. For this reason, the study of genetic variation within species is of importance for their epidemiological implication. We employed two SNP genotyping technologies to distinguish G1 and G3 ). 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 genotyping.
（）是动物和人类囊性棘球蚴病的病原体。不同基因型在其生命周期、宿主选择性和致病性方面表现出极大的多样性。因此，研究物种内的基因变异对其流行病学意义至关重要。我们采用了两种单核苷酸多态性（SNP）基因分型技术来区分G1和G3（）基因型。通过对线粒体nad5基因片段进行扩增和测序，鉴定了从不同动物物种的包虫囊肿中提取的DNA样本（n = 28）的基因型。针对已知能够区分G1和G3的三个nad5信息位点（SNP758、1123和1380），开发了两种SYBR Green和三种TaqMan实时荧光定量PCR检测方法。基于循环阈值（Ct）并结合熔解温度（Tm）分析的SYBR Green PCR基因分型方法，对SNP1123和SNP1380进行检测时，未能鉴定出一个DNA样本。针对SNP758、1123和1380的TaqMan检测方法有效地证实了通过桑格测序获得的基因型鉴定结果。我们的结果表明，本研究中开发的三种TaqMan检测方法相结合，是一种用于基因分型的、有价值且具有成本效益的替代DNA测序的工具。
