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

• Published in: Journal of visualized experiments 2016-03 (109)
• Main Authors: Hue, Erika S, Fortier, Christine I, Laurent, Aurélie M, Quesnelle, Yann F, Fortier, Guillaume D, Legrand, Loïc J, Pronost, Stephane L
• Format: Article
• Language: English
• Subjects: Animal biology; Animals; Bronchoalveolar Lavage Fluid - virology; Herpesviridae Infections - diagnosis; Herpesviridae Infections - genetics; Horse Diseases - diagnosis; Horse Diseases - genetics; Horses; Immunology; Life Sciences; 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; Veterinary medicine and animal Health; Viral Load - genetics
• ISSN: 1940-087X; 1940-087X
• DOI: 10.3791/53672

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.