Piorkowski G., Baronti Cécile, de Lamballerie Xavier, de Fabritus L., Bichaud L., Pastorino B. A., Bessaud M. (2014). Development of generic Taqman PCR and RT-PCR assays for the detection of DNA and mRNA of beta-actin-encoding sequences in a wide range of animal species. Journal of Virological Methods, 202, p. 101-105. ISSN 0166-0934.
Titre du document
Development of generic Taqman PCR and RT-PCR assays for the detection of DNA and mRNA of beta-actin-encoding sequences in a wide range of animal species
Piorkowski G., Baronti Cécile, de Lamballerie Xavier, de Fabritus L., Bichaud L., Pastorino B. A., Bessaud M.
Source
Journal of Virological Methods, 2014,
202, p. 101-105 ISSN 0166-0934
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 beta-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 beta-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 beta-actin-encoding sequences as endogenous control. This assay will constitute a precious tool for virologists working with multiple cell lines or animal models.
Plan de classement
Sciences fondamentales / Techniques d'analyse et de recherche [020]
;
Entomologie médicale / Parasitologie / Virologie [052]
