Sensitive and accurate DNA metabarcoding of parasitic helminth mock communities using the mitochondrial rRNA genes

Abstract

Next-generation sequencing technologies have accelerated the pace of helminth DNA metabarcoding research, enabling species detection in bulk community samples. However, finding suitable genetic markers with robust species-level resolution and primers targeting a broad species range among parasitic helminths are some of the challenges faced. This study aimed to demonstrate the potential use of the mitochondrial 12S and 16S rRNA genes for parasitic helminth (nematodes, trematodes, cestodes) DNA metabarcoding. To demonstrate the robustness of the 12S and 16S rRNA genes for DNA metabarcoding, we determined the proportion of species successfully recovered using mock helminth communities without environment matrix and mock helminth communities artificially spiked with environmental matrices. The environmental matrices are human fecal material, garden soil, tissue, and pond water. Our results revealed the robustness of the mitochondrial rRNA genes, through the high sensitivity of the 12S rRNA gene, and the effectiveness of the 12S and 16S primers targeting platyhelminths. With the mitochondrial rRNA genes, a broad range of parasitc helminths were successfully detected to the species level. The potential of the mitochondrial rRNA genes for helminth DNA metabarcoding was demonstrated, providing a valuable gateway for future helminth DNA metabarcoding applications like helminth detection and biodiversity studies.

A molecular assessment of Ostertagia leptospicularis and Spiculopteragia asymmetrica among wild fallow deer in Northern Ireland and implications for false detection of livestock-associated species

“…PCR-based amplification yields fluorescence from specific probes in loop-mediated isothermal amplification (LAMP) [ 52 , 56 ], Q-PCR [ 54 , 68 , 77 ] and ddPCR [ 78 ], size-specific products in endpoint PCR and semi-quantitative PCR [ 48 , 49 ] or products with specific melt curves in high-resolution melt curve analyses [ 48 , 55 ]. Molecular monitoring of GINs can utilize a number of genetic sources such as: (1) adult worms from necropsies [ 55 , 79 ], (2) faecal DNA [ 48 , 79 ], (3) isolated eggs from faeces [ 77 ], (4) cultured larvae [ 53 , 57 ], (5) pasture larvae [ 80 ] and (6) environmental DNA [ 81 ]. However, validation of probe specificity has typically not taken wildlife species into consideration, although multiple studies have been mindful to detect wildlife species [ 39 , 46 , 59 , 60 ].…”

From past to present: opportunities and trends in the molecular detection and diagnosis of Strongyloides stercoralis

“…Illumina sequencing metagenomics were used to detect S. stercoralis in CSF and bronchoalveolar lavage fluid samples from patients, showing the high sensitivity of the technique and potential for use [ 48 – 50 ]. Additionally, targeted amplicon Illumina sequencing of the 12S and 16S rRNA genes through DNA metabarcoding has also demonstrated the potential of detecting S. stercoralis larvae spiked in mock helminth communities and environment matrices [ 51 ]. Although conventional molecular-based methods are still popular, the shift toward NGS is certain in the future.…”

ABIapp: A user-friendly tool for precise parasitic helminth species delimitation

“…PCR was conducted in a final volume of 30 μl, comprising 15 μl of 2X i-TaqTM mastermix (iNtRON Biotechnology, Gyeonggi, South Korea), 10 μM to 50 μM of each primer, and the template DNA. We adhered to the thermocycling conditions specified in the publications introducing these primers (Bowles et al, 1995;Callejón et al, 2013;Chan, Chaisiri, Morand, et al, 2020;Chan et al, 2022;Holterman et al, 2006;Routtu et al, 2014). We visualized amplicons on a 1% agarose gel stained with RedSafe® (Thomas Scientific, New Jersey, USA).…”