A one-step SYBR Green I-based product-enhanced reverse transcriptase assay for the quantitation of retroviruses in cell culture supernatants.
PCR-enhanced reverse transcriptase assays (PERT) are sensitive tools for the detection of retroviruses in biological samples. The adaptation of real-time PCR techniques based on fluorescent probes (F-PERT) has added a reliable quantitative capacity to the assay. In the interest of economy and time, the SYBR Green I-based real-time detection system was used to establish a convenient one-step PERT assay (SG-PERT). This assay can be completed in 2h, is linear over six orders of magnitude and can be used to quantify retroviruses belonging to divergent species, such as the human immunodeficiency virus type 1 (HIV-1), murine leukemia virus (MLV) and prototypic foamy virus (PFV).
Pizzato
Massimo
M
Jefferiss Trust Laboratories, Wright-Fleming Institute, Imperial College, London W2 1PG, United Kingdom. m.pizzato@imperial.ac.uk
Erlwein
Otto
O
Bonsall
David
D
Kaye
Stephen
S
Muir
David
D
McClure
Myra O
MO
D052160:Benzothiazoles; D018929:Cell Culture Techniques; D002460:Cell Line; D003959:Diamines; D005456:Fluorescent Dyes / Q000737:chemistry*; D015497:HIV-1 / Q000302:isolation & purification; D006801:Humans; D009930:Organic Chemicals / Q000737:chemistry*; D011804:Quinolines; D012367:RNA, Viral / Q000032:analysis; D015203:Reproducibility of Results; D012190:Retroviridae / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016092:Spumavirus / Q000302:isolation & purification
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I