Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis.
Abstract: Strongyle and trichostrongyle parasites are ubiquitous nematodes of grazing livestock. Several molecular diagnostic tests are based upon measuring and quantifying DNA obtained from parasite eggs. It is well known that such eggs undergo development during storage, but it remains unknown to which extent developmental stages can affect the variation of diagnostic test results. This study investigated the influence of developmental stages of strongyle eggs on the variation real-time polymerase chain reaction (PCR) results. Mixed species strongyle eggs were obtained from the faeces of a naturally infected horse. Eggs were isolated and placed in microtiter plates with demineralized water. A total of 25 wells containing 100 eggs each were set up and kept refrigerated for up to five days. Once daily, five wells were examined on an inverted microscope at 100× magnification, where the developmental stages of the eggs were noted, and then eggs harvested for DNA extraction. The protocol was repeated three times. Genomic DNA was extracted using a commercial kit previously validated for strongyle type eggs. PCR reactions were performed with a primer set specific for the ribosomal DNA region for all strongyle type parasites (NC1, NC2). SYBR Green Real-Time PCRs were performed in triplicates. Results revealed a statistically significant increase in PCR yield after three days, which was statistically associated with beginning embryonation of the eggs. In conclusion, storage time and developmental stage of strongyle eggs are significant sources of error in studies based on quantitative real-time PCR analysis. This study suggests that for refrigerated storage of more than three days, eggs should be inactivated and preserved for further analysis.
Copyright © 2012 Elsevier B.V. All rights reserved.
Publication Date: 2012-09-05 PubMed ID: 23009948DOI: 10.1016/j.vetpar.2012.08.018Google Scholar: Lookup
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- Journal Article
Summary
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The research article discusses how the developmental stages of a common type of parasite found in livestock, the Strongyle, can impact the diagnostic results of DNA-based tests. The developmental stages of the parasite eggs specifically affect the results from a common DNA test, the real-time Polymerase Chain Reaction (PCR).
Background
- The study is centered on strongyle and trichostrongyle parasites, which are nematodes commonly found in the digestive system of grazing livestock.
- DNA testing is often used to diagnose the presence of these parasites by measuring and quantifying the DNA obtained from their eggs.
- It is known that the eggs of these parasites continue to develop during storage, but the extent to which this development affects diagnostic results was previously unknown.
Methods and Experimentation
- The researchers collected strongyle eggs from the feces of a naturally infected horse.
- These eggs were then isolated and kept in demineralized water for up to five consecutive days within a microtiter plate, with each well containing a set amount of 100 eggs.
- Each day, the researchers examined five randomized wells using an inverted microscope, noting the developmental stages of the eggs before harvesting them for DNA extraction.
- They pulled the extracted DNA through a polymerase chain reaction (PCR), utilizing specific primers for strongyle type parasites.
- The team repeated this experiment three times for reliability.
Findings and Conclusion
- The researchers found a significant increase in PCR yield after three days of refrigeration, which was associated with the beginning of embryonation (the early developmental stage) of the eggs.
- This suggests that both storage time and developmental stage of the parasites’ eggs play an influential role in the outcomes of PCR diagnostic tests.
- To mitigate the error, the researchers advise that if the eggs are to be refrigerated for a time period of more than three days, they should be inactivated and preserved for later analysis.
Cite This Article
APA
Andersen UV, Haakansson IT, Roust T, Rhod M, Baptiste KE, Nielsen MK.
(2012).
Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis.
Vet Parasitol, 191(1-2), 191-196.
https://doi.org/10.1016/j.vetpar.2012.08.018 Publication
Researcher Affiliations
- Department of Large Animal Sciences, University of Copenhagen, Taastrup, Denmark. ullava@sund.ku.dk
MeSH Terms
- Animals
- Cold Temperature
- DNA, Helminth / genetics
- Feces / parasitology
- Horse Diseases / diagnosis
- Horses
- Real-Time Polymerase Chain Reaction / standards
- Real-Time Polymerase Chain Reaction / veterinary
- Strongyle Infections, Equine / diagnosis
- Strongylida / growth & development
- Strongylida / physiology
- Time Factors
Citations
This article has been cited 2 times.- Papaiakovou M, Wright J, Pilotte N, Chooneea D, Schär F, Truscott JE, Dunn JC, Gardiner I, Walson JL, Williams SA, Littlewood DTJ. Pooling as a strategy for the timely diagnosis of soil-transmitted helminths in stool: value and reproducibility.. Parasit Vectors 2019 Sep 16;12(1):443.
- Kaspar A, Pfister K, Nielsen MK, Silaghi C, Fink H, Scheuerle MC. Detection of Strongylus vulgaris in equine faecal samples by real-time PCR and larval culture - method comparison and occurrence assessment.. BMC Vet Res 2017 Jan 11;13(1):19.
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