title: Development of generic Taqman PCR and RT-PCR assays for the detection of DNA and mRNA of β-actin-encoding sequences in a wide range of animal species.
abstract: As a member of the European Virus Archive (EVA) consortium, our laboratory is developing and maintaining a large collection of viruses. This collection implies the use of a panel of cell lines originating from various animal species. In order to make easier the handling of such a large panel of cell lines, wide spectrum real-time PCR and RT-PCR assays were developed to allow the detection and the quantification of DNA and mRNA of β-actin, one of the most commonly used eukaryotic housekeeping genes. By using two degenerated primers and a unique probe, these two assays were shown to detect nucleic acids of a panel of vertebrate and invertebrate cell lines commonly used in animal virology. This panel included human, monkey, rodent, dog, pig, fish, batrachian, mosquito and tick cell lines. Additionally, the two assays amplified successfully β-actin-encoding sequences of sandflies. Sensitivity evaluation performed on synthetic DNA and RNA sequences showed that the two assays were very sensitive and suitable for accurate quantification. The two assays constitute together a convenient method suitable for multiple purposes. They can be used for instance to estimate the amount of contaminating cellular genetic material prior to sequence-independent amplification of viral genomes achieved before high-throughput sequencing, to evaluate the efficiency of DNase and/or RNase treatments performed on cellular extract and to check nucleic acid extraction by using β-actin-encoding sequences as endogenous control. This assay will constitute a precious tool for virologists working with multiple cell lines or animal models.
authors.affiliation: UMR 190 "Emergence des Pathologies Virales", Aix-Marseille Univ., IRD French Institute of Research for Development, EHESP French School of Public Health, Marseille, France.
authors.forename: Géraldine
authors.initials: G
authors.lastname: Piorkowski
authors.affiliation: UMR 190 "Emergence des Pathologies Virales", Aix-Marseille Univ., IRD French Institute of Research for Development, EHESP French School of Public Health, Marseille, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Baronti
authors.affiliation: UMR 190 "Emergence des Pathologies Virales", Aix-Marseille Univ., IRD French Institute of Research for Development, EHESP French School of Public Health, Marseille, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: de Lamballerie
authors.affiliation: UMR 190 "Emergence des Pathologies Virales", Aix-Marseille Univ., IRD French Institute of Research for Development, EHESP French School of Public Health, Marseille, France.
authors.forename: Lauriane
authors.initials: L
authors.lastname: de Fabritus
authors.affiliation: UMR 190 "Emergence des Pathologies Virales", Aix-Marseille Univ., IRD French Institute of Research for Development, EHESP French School of Public Health, Marseille, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Bichaud
authors.affiliation: UMR 190 "Emergence des Pathologies Virales", Aix-Marseille Univ., IRD French Institute of Research for Development, EHESP French School of Public Health, Marseille, France.
authors.forename: Boris A
authors.initials: BA
authors.lastname: Pastorino
authors.affiliation: UMR 190 "Emergence des Pathologies Virales", Aix-Marseille Univ., IRD French Institute of Research for Development, EHESP French School of Public Health, Marseille, France. Electronic address: bessaud@laposte.net.
authors.forename: Maël
authors.initials: M
authors.lastname: Bessaud
mesh_terms: D000199:Actins / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D018070:Biological Specimen Banks; D002460:Cell Line; D004247:DNA / Q000032:analysis* / Q000235:genetics; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014773:Virology / Q000379:methods*
chemical_list: D000199:Actins; D012333:RNA, Messenger; D004247:DNA
keywords: ACTB gene; Actin; Housekeeping gene; Next generation sequencing; Taqman assay