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Highly sensitive SIV plasma viral load assay: practical considerations, realistic performance expectations, and application to reverse engineering of vaccines for AIDS
Author(s) -
Nichole Cline A.,
Bess Julian W.,
Piatak Michael, Jr,
Lifson Jeffrey D.
Publication year - 2005
Publication title -
journal of medical primatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.31
H-Index - 42
eISSN - 1600-0684
pISSN - 0047-2565
DOI - 10.1111/j.1600-0684.2005.00128.x
Subject(s) - viral load , simian immunodeficiency virus , reverse transcriptase , virology , virus , biology , viral replication , reverse transcription polymerase chain reaction , real time polymerase chain reaction , human immunodeficiency virus (hiv) , sensitivity (control systems) , polymerase chain reaction , messenger rna , genetics , gene , electronic engineering , engineering
As new assay methods for quantitative reverse transcription‐polymerase chain reaction (RT‐PCR), such as real time RT‐PCR techniques, approach theoretical limits of per reaction sensitivity, further increments in the sensitivity of measurements of viral load can only be achieved by increasing the amount of input RNA per reaction. We describe a robust, convenient, rapid integrated approach for specimen preparation and real time RT‐PCR assay for plasma simian immunodeficiency virus (SIV) RNA viral load that provides a threshold sensitivity of 10 copy Eq/ml, and tolerates less than optimally processed specimens. The method provides accurate quantitation of viral load for the SIV virus isolates in common use for non‐human primate studies. We demonstrate the utility of the method in sensitively tracking viral load in an animal showing effective control of viral replication to levels below the threshold for quantitation in conventional assays.