Abstract
Introduction 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. Materials and methods 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 Cryptosporidium parvum 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 C. hominis, C. muris, Giardia duodenalis and Toxoplasma gondii individually or together. Results 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 C. parvum 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 C. parvum, C. hominis, C. muris, G. duodenalis and T. gondii . Discussion Our metagenomic procedure led to the identification of C. parvum 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.
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Claims 5
Claim · model-written summary
The mNGS assay identified as few as 100 oocysts of C. parvum in 25 g of fresh lettuce.
- Taxon
- fresh lettucee2
- Interactor
- C. parvum
Cryptosporidium parvume2other - Outcome
- identificatione2
- Value
- 100 oocystse2
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Lettuce (25 g) was spiked with varying numbers of Cryptosporidium parvum oocysts”
e2“Nanopore sequencing followed by bioinformatic analysis led to the consistent identification of as few as 100 oocysts of C. parvum in 25 g of fresh lettuce”
Claim · model-written summary
Ion S5 sequencing gave results similar to Nanopore sequencing for the mNGS assay on lettuce.
- Taxon
- lettuce
Lactuca sativae2 - Outcome
- Similar resultse3
- Qualifier
- Ion S5 sequencing platforme3
- Comparator
- Nanopore sequencinge2
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Lettuce (25 g) was spiked with varying numbers of Cryptosporidium parvum oocysts”
e2“Nanopore sequencing followed by bioinformatic analysis led to the consistent identification of as few as 100 oocysts of C. parvum in 25 g of fresh lettuce”
e3“Similar results were obtained using the Ion S5 sequencing platform”
Claim · model-written summary
The assay simultaneously detected C. parvum and G. duodenalis on lettuce, among other protozoa.
- Taxon
- LettuceLactuca sativae1
- Interactor
- C. parvumCryptosporidium parvume4other
- Second interactor
- G. duodenalis
Giardia duodenalise4other - Outcome
- simultaneous detectione4
- Qualifier
- C. hominis, C. muris, G. duodenalis and T. gondiie4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Lettuce (25 g) was spiked with varying numbers of Cryptosporidium parvum oocysts”
e4“The assay proved useful for the simultaneous detection of C. parvum, C. hominis, C. muris, G. duodenalis and T. gondii”
Claim · model-written summary
Amplification gave 0.16–8.25 μg of DNA from lettuce washes, sufficient to perform mNGS.
- Taxon
- LettuceLactuca sativae1
- Outcome
- generatione5
- Value
- 0.16–8.25 μge5
- Qualifier
- sufficient to perform mNGSe5
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Lettuce (25 g) was spiked with varying numbers of Cryptosporidium parvum oocysts”
e5“Amplification of extracted DNA led to the generation of 0.16–8.25 μg of DNA (median = 4.10 μg), sufficient to perform mNGS”
Claim · model-written summary
Lysis of oocysts and cysts was rapidly achieved within 3 min on lettuce, a prerequisite for sensitive detection.
- Taxon
- LettuceLactuca sativae1
- Outcome
- lysis of oocysts and cystse6
- Value
- within 3 mine6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Lettuce (25 g) was spiked with varying numbers of Cryptosporidium parvum oocysts”
e6“The efficient lysis of oocysts and cysts was a prerequisite for the sensitive detection of parasite DNA and was rapidly achieved within 3 min”