Quantitative determination of lentiviral vector particle numbers by real-time PCR.
Here, we describe a quantitative, DNA-based, real-time PCR approach to determine the number of lentivirus particles that are present in vector preparations. In this approach, the minus strong-stop cDNA fragment that is present in viral capsids serves as template for PCR. Using this technology, we found that only 0.1%-1% of the virus particles that are present in vector preparations are infectious. The approach described here is rapid, reliable, and simple in concept and can be used to estimate both vector particles in supernatants and the number of infectious particles. Also, this approach can easily be adapted to a high-throughput system by using 96-well plates and a 2-h running time.
Scherr
M
M
Medizinische Hochschule Hannover, Abt. Hämatologie u. Onkologie, Germany. scherr.michaela@mh-hannover.de
Battmer
K
K
Blömer
U
U
Ganser
A
A
Grez
M
M
D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D014162:Transfection; D014771:Virion / Q000235:genetics / Q000302:isolation & purification*
D008164:Luminescent Proteins; D012367:RNA, Viral; D049452:Green Fluorescent Proteins