Naushad Sohail, Gao Ruimin, Duceppe Marc-Olivier, Dupras Andree Ann, Reiling Sarah J, Merks Harriet, Dixon Brent, Ogunremi Dele
Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency, Ottawa, ON, Canada.
Bureau of Microbial Hazards, Food and Nutrition Directorate, Health Canada, Ottawa, ON, Canada.
Front Microbiol. 2025 Mar 14;16:1566579. doi: 10.3389/fmicb.2025.1566579. eCollection 2025.
Infections with protozoan parasites associated with the consumption of fresh produce is an on-going issue in developed countries but mitigating the risk is hampered by the lack of adequate methods for their detection and identification.
We developed a metagenomic next-generation sequencing (mNGS) assay using a MinION sequencer for the identification of parasites in intentionally contaminated lettuce to achieve a more accurate and rapid method than the traditional molecular and microscopy methods commonly used for regulatory purposes. Lettuce (25 g) was spiked with varying numbers of oocysts, and microbes washed from the surface of the lettuce were lysed using the OmniLyse device. DNA was then extracted by acetate precipitation, followed by whole genome amplification. The amplified DNA was sequenced by nanopore technology and validated with the Ion Gene Studio S5, and the generated fastq files raw reads were uploaded to the CosmosID webserver for the bioinformatic identification of microbes in the metagenome. To demonstrate the ability of the procedure to distinguish other common food and waterborne protozoan parasites, lettuce was also spiked with and individually or together.
The efficient lysis of oocysts and cysts was a prerequisite for the sensitive detection of parasite DNA and was rapidly achieved within 3 min. Amplification of extracted DNA led to the generation of 0.16-8.25 μg of DNA (median = 4.10 μg), sufficient to perform mNGS. Nanopore sequencing followed by bioinformatic analysis led to the consistent identification of as few as 100 oocysts of in 25 g of fresh lettuce. Similar results were obtained using the Ion S5 sequencing platform. The assay proved useful for the simultaneous detection of and .
Our metagenomic procedure led to the identification of present on lettuce at low numbers and successfully identified and differentiated other protozoa either of the same genus or of different genera. This novel mNGS assay has the potential for application as a single universal test for the detection of foodborne parasites, and the subtyping of parasites for foodborne outbreak investigations and surveillance studies.
食用新鲜农产品引发的原生动物寄生虫感染在发达国家一直是个问题，但由于缺乏足够的检测和鉴定方法，降低风险的工作受到阻碍。
我们开发了一种宏基因组下一代测序（mNGS）检测方法，使用MinION测序仪鉴定故意污染生菜中的寄生虫，以获得一种比监管目的常用的传统分子和显微镜方法更准确、快速的方法。将不同数量的卵囊接种到25克生菜中，用OmniLyse设备裂解从生菜表面冲洗下来的微生物。然后通过醋酸盐沉淀提取DNA，接着进行全基因组扩增。扩增后的DNA通过纳米孔技术测序，并用Ion Gene Studio S5进行验证，生成的fastq文件原始读数上传到CosmosID网络服务器，用于宏基因组中微生物的生物信息学鉴定。为了证明该方法区分其他常见食源和水源性原生动物寄生虫的能力，生菜还分别或同时接种了 和 。
卵囊和包囊的有效裂解是灵敏检测寄生虫DNA的前提，在3分钟内迅速实现。提取DNA的扩增产生了0.16 - 8.25微克DNA（中位数 = 4.10微克），足以进行mNGS。纳米孔测序后进行生物信息学分析，能够一致鉴定出25克新鲜生菜中低至100个 的卵囊。使用Ion S5测序平台也得到了类似结果。该检测方法被证明可用于同时检测 和 。
我们的宏基因组方法能够鉴定出生菜上数量较少的 ，并成功鉴定和区分了同一属或不同属的其他原生动物。这种新型mNGS检测方法有潜力作为一种单一通用检测方法应用于食源寄生虫的检测，以及用于食源性疾病暴发调查和监测研究中的寄生虫亚型分析。
