High-throughput real-time PCR for early detection and quantitation of arenavirus Tacaribe.
Arenaviruses merit significant attention both as causative agents of endemic hemorrhagic fevers and as model systems to study the immune response to acute and persistent viral infections. Development of highly sensitive quantitative screening methods to detect arenavirus is critical for early diagnosis of patients, to screen the rodent population in endemic areas, and as a research tool to confirm effective tissue clearance during the development of anti-viral strategies. This study describes a novel sensitive and reproducible method to quantify prototypic new world arenavirus Tacaribe RNA in cell cultures and tissues using a real-time TaqMan PCR-based detection system. The method has a sensitivity of 100 RNA copies per 200ng of total RNA, making it 2 logs more sensitive than the currently utilized TCID(50) method, and a linear range from 10(2) to 10(9) copies/reaction. The qRT-PCR method is high-throughput and screening can be achieved in <2h allowing for diagnosis of infected patients before the onset of symptoms. This new method is a powerful tool to screen populations for infection and monitor the clearance achieved by available therapies, and serves as a model diagnostic tool for other arenaviruses.
Grajkowska
Lucja T
LT
Division of Therapeutic Proteins, Office of Biotechnology Products, CDER, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, USA.
Pedras-Vasconcelos
Joao A
JA
Sauder
Christian
C
Verthelyi
Daniela
D
Puig
Montserrat
M
D000818:Animals; D006477:Arenaviruses, New World / Q000302:isolation & purification*; D002522:Chlorocebus aethiops; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics*; D012680:Sensitivity and Specificity; D014709:Vero Cells
D012367:RNA, Viral