Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis

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Standard

Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis. / Andersen, Ulla Vestergaard; Haakansson, I. T.; Roust, Tina; Rhod, Maria; Baptiste, Keith Edward; Nielsen, Martin Krarup.

I: Veterinary Parasitology, Bind 191, Nr. 1-2, 2013, s. 191-196.

Publikation: Bidrag til tidsskriftTidsskriftartikelfagfællebedømt

Harvard

Andersen, UV, Haakansson, IT, Roust, T, Rhod, M, Baptiste, KE & Nielsen, MK 2013, 'Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis', Veterinary Parasitology, bind 191, nr. 1-2, s. 191-196. https://doi.org/10.1016/j.vetpar.2012.08.018

APA

Andersen, U. V., Haakansson, I. T., Roust, T., Rhod, M., Baptiste, K. E., & Nielsen, M. K. (2013). Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis. Veterinary Parasitology, 191(1-2), 191-196. https://doi.org/10.1016/j.vetpar.2012.08.018

Vancouver

Andersen UV, Haakansson IT, Roust T, Rhod M, Baptiste KE, Nielsen MK. Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis. Veterinary Parasitology. 2013;191(1-2):191-196. https://doi.org/10.1016/j.vetpar.2012.08.018

Author

Andersen, Ulla Vestergaard ; Haakansson, I. T. ; Roust, Tina ; Rhod, Maria ; Baptiste, Keith Edward ; Nielsen, Martin Krarup. / Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis. I: Veterinary Parasitology. 2013 ; Bind 191, Nr. 1-2. s. 191-196.

Bibtex

@article{6cef7e60650f4b6d83a605dbab191cf9,
title = "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.",
keywords = "Animals, Cold Temperature, DNA, Helminth, Feces, Horse Diseases, Horses, Real-Time Polymerase Chain Reaction, Strongyle Infections, Equine, Strongylida, Time Factors",
author = "Andersen, {Ulla Vestergaard} and Haakansson, {I. T.} and Tina Roust and Maria Rhod and Baptiste, {Keith Edward} and Nielsen, {Martin Krarup}",
note = "Copyright {\textcopyright} 2012 Elsevier B.V. All rights reserved.",
year = "2013",
doi = "10.1016/j.vetpar.2012.08.018",
language = "English",
volume = "191",
pages = "191--196",
journal = "Veterinary Parasitology",
issn = "0304-4017",
publisher = "Elsevier",
number = "1-2",

}

RIS

TY - JOUR

T1 - Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis

AU - Andersen, Ulla Vestergaard

AU - Haakansson, I. T.

AU - Roust, Tina

AU - Rhod, Maria

AU - Baptiste, Keith Edward

AU - Nielsen, Martin Krarup

N1 - Copyright © 2012 Elsevier B.V. All rights reserved.

PY - 2013

Y1 - 2013

N2 - 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.

AB - 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.

KW - Animals

KW - Cold Temperature

KW - DNA, Helminth

KW - Feces

KW - Horse Diseases

KW - Horses

KW - Real-Time Polymerase Chain Reaction

KW - Strongyle Infections, Equine

KW - Strongylida

KW - Time Factors

U2 - 10.1016/j.vetpar.2012.08.018

DO - 10.1016/j.vetpar.2012.08.018

M3 - Journal article

C2 - 23009948

VL - 191

SP - 191

EP - 196

JO - Veterinary Parasitology

JF - Veterinary Parasitology

SN - 0304-4017

IS - 1-2

ER -

ID: 45155989