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Journal of visualized experiments : JoVE2016; (109); 53672; doi: 10.3791/53672

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids.

Abstract: The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.
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Publication Date: 2016-03-17 PubMed ID: 27022998PubMed Central: PMC4829031DOI: 10.3791/53672Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article describes the development and validation of a quantitative RT-PCR method for the detection and quantification of Equid Herpesvirus-2 (EHV-2) in equine respiratory fluids. The process involved two distinct steps of characterization, and also highlighted the importance of validating all intervening steps for an accurate determination of the viral genome load.

Development and First Validation Step

  • The researchers designed a quantitative RT-PCR protocol for detecting and quantifying EHV-2 in equine respiratory fluids. This technique is primarily based on the NF U47-600 norm, a recognized standard in the field of molecular diagnostics.
  • The initial phase involved the development and a preliminary validation of the proposed method.

Characterization Steps

  • The procedure incorporated two distinct characterization steps in accordance with the standards defined by the AFNOR norm.
  • The first step focused on the characterization of the qRT-PCR assay in isolation, while the second pertained to the characterization of the whole analytical method, including each step from nucleic acid extraction up to PCR analysis.

Need for Comprehensive Validation

  • The paper underscores the necessity of conducting a thorough validation of the entire analytical method for accurate virus detection through qRT-PCR and precise determination of viral genome load.
  • The extraction step is typically the major point of loss of biological material, and therefore, is regarded as a key source of error in quantification. Given this, the researchers stress on the need to incorporate the range of plasmid dilution prior to the extraction step as recommended by the AFNOR norm NF-U-47-600.

Viral Genome Load and Interpretation of Results

  • The limits of quantification are influenced by the source from which the virus is extracted.
  • The presented results are expressed in International Units (IU) to facilitate easy comparison between different laboratories. This common unit of measurement is helpful in standardizing result interpretation across various labs.

Contribution to the Field

  • This newly developed method for the characterization of qRT-PCR can significantly aid in harmonizing data presentation and interpretation among different laboratories, thereby contributing to more accurate and reliable diagnostics of EHV-2 in the field of equine health.

Cite This Article

APA
Hue ES, Fortier CI, Laurent AM, Quesnelle YF, Fortier GD, Legrand LJ, Pronost SL. (2016). Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids. J Vis Exp(109), 53672. https://doi.org/10.3791/53672

Publication

Journal of visualized experiments : JoVE
ISSN: 1940-087X
NlmUniqueID: 101313252
Country: United States
Language: English
Issue: 109
PII: 53672

Researcher Affiliations

Hue, Erika S
  • LABÉO Frank Duncombe; Unité de Risques Microbiens (U2RM, EA 4655), Normandy University; Hippolia Foundation.
Fortier, Christine I
  • LABÉO Frank Duncombe; Hippolia Foundation.
Laurent, Aurélie M
  • LABÉO Frank Duncombe.
Quesnelle, Yann F
  • LABÉO Frank Duncombe.
Fortier, Guillaume D
  • LABÉO Frank Duncombe; Unité de Risques Microbiens (U2RM, EA 4655), Normandy University; Hippolia Foundation.
Legrand, Loïc J
  • LABÉO Frank Duncombe; Unité de Risques Microbiens (U2RM, EA 4655), Normandy University; Hippolia Foundation.
Pronost, Stephane L
  • LABÉO Frank Duncombe; Unité de Risques Microbiens (U2RM, EA 4655), Normandy University; Hippolia Foundation; Stephane.Pronost@calvados.fr.

MeSH Terms

  • Animals
  • Bronchoalveolar Lavage Fluid / virology
  • Herpesviridae Infections / diagnosis
  • Herpesviridae Infections / genetics
  • Horse Diseases / diagnosis
  • Horse Diseases / genetics
  • Horses
  • Plasmids / analysis
  • Plasmids / genetics
  • Real-Time Polymerase Chain Reaction / methods
  • Real-Time Polymerase Chain Reaction / standards
  • Reproducibility of Results
  • Rhadinovirus / genetics
  • Rhadinovirus / isolation & purification
  • Tumor Virus Infections / diagnosis
  • Tumor Virus Infections / genetics
  • Viral Load / genetics

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Citations

This article has been cited 6 times.
  1. Lee MA, Shen Z, Holcombe HR, Ge Z, Franklin EG, Ricart Arbona RJ, Lipman NS, Fox JG, Sheh A. Detection of Myocoptes musculinus in Fur Swab and Fecal Samples by Using PCR Analysis. J Am Assoc Lab Anim Sci 2019 Nov 1;58(6):796-801.
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