Cort-pcr assay for the discrimination of endogenous reverse...

C - Chemistry – Metallurgy – 12 – Q

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C12Q 1/70 (2006.01) C12Q 1/68 (2006.01)

Patent

CA 2376535

The present invention relates to the detection of the presence of reverse transcriptase enzymatic activity in eukary-otic cell lines. Specifically, the present invention comprises a method for discriminating between endogenous reverse transcriptase activity in a substrate cell line (e.g., avian) used as substrates for the manufacture of biological products (including viral vaccines) from exogenous infectious retrovirus by using a combination of transmissibility assay (viral replication) and PBRT assay (RT-PCR). Appropriate levels of viral amplification and number of PCR cycles are determined and employed that permit detection of exogenous infectious RT activity but not endogenous RT activity.

La présente invention concerne la détection de l'activité enzymatique transcriptase inverse dans des lignées de cellules eucaryotes. Spécifiquement, cette invention concerne un procédé de discrimination entre l'activité transcriptase inverse endogène dans une lignée cellulaire substrat (par exemple, aviaire) utilisée comme substrats pour la fabrication de produits biologiques (y compris des vaccins viraux) et le rétrovirus infectieux exogène, en utilisant une combinaison de dosage de facteur de transmission (réplication virale) et le dosage PBRT (RT-PCR). Des niveaux appropriés d'amplification virale et de nombres de cycles PCR sont déterminés et utilisés pour permettre la détection de l'activité RT infectieuse exogène mais pas de l'activité RT endogène.

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