Bailly Eloïse, Valot Stéphane, Vincent Anne, Duffourd Yannis, Grangier Nadège, Chevarin Martin, Costa Damien, Razakandrainibe Romy, Favennec Loïc, Basmaciyan Louise, Dalle Frédéric
Parasitology-Mycology Laboratory, University Hospital Biology Platform, Dijon University Hospital Center, 21000 Dijon, France.
Associated Laboratory CNR-LE for Cryptosporidiosis, University Hospital Biology Platform, Dijon University Hospital Center, 21000 Dijon, France.
Pathogens. 2022 Aug 18;11(8):938. doi: 10.3390/pathogens11080938.
. Nowadays, most of the and epidemiological studies are based on gp60 gene subtyping using the Sanger sequencing (SgS) method. Unfortunately, SgS presents the limitation of being unable to detect mixed infections. Next-Generation Sequencing (NGS) seems to be an interesting solution to overcome SgS limits. Thus, the aim of our study was to (i) evaluate the reliability of NGS as a molecular typing tool for cryptosporidiosis, (ii) investigate the genetic diversity of the parasite and the frequency of mixed infections, (iii) assess NGS usefulness in sp. outbreak investigations, and (iv) assess an interpretation threshold of sequencing data. . 108 DNA extracts from positive samples were sequenced by NGS. Among them, two samples were used to validate the reliability of the subtyping obtained by NGS and its capacity to detect DNA mixtures. In parallel, 106 samples from French outbreaks were used to expose NGS to epidemic samples. . NGS proved suitable for sp. subtyping at the gp60 gene locus, bringing more genetic information compared to SgS, especially by working on many samples simultaneously and detecting more diversity. . This study confirms the usefulness of NGS applied to and epidemiological studies, especially aimed at detecting minority variants.
如今，大多数隐孢子虫和微小隐孢子虫的流行病学研究都是基于使用桑格测序（SgS）方法对gp60基因进行亚型分型。遗憾的是，SgS存在无法检测混合感染的局限性。新一代测序（NGS）似乎是克服SgS局限性的一个有趣解决方案。因此，我们研究的目的是：（i）评估NGS作为隐孢子虫病分子分型工具的可靠性；（ii）调查该寄生虫的遗传多样性和混合感染频率；（iii）评估NGS在微小隐孢子虫爆发调查中的实用性；（iv）评估测序数据的解读阈值。108份阳性样本的DNA提取物通过NGS进行测序。其中，两个样本用于验证通过NGS获得的亚型分型的可靠性及其检测DNA混合物的能力。同时，来自法国疫情爆发的106个样本用于让NGS处理疫情样本。NGS被证明适用于微小隐孢子虫在gp60基因位点的亚型分型，与SgS相比能提供更多遗传信息，特别是通过同时处理多个样本并检测到更多的多样性。这项研究证实了NGS应用于隐孢子虫和微小隐孢子虫流行病学研究的实用性，尤其是旨在检测少数变异体。
