Development of a real time quantitative PCR assay for detection of porcine endogenous retrovirus.
Real time PCR technology was applied to the development of assays for detection and quantitation of porcine endogenous retrovirus (PERV) RNA and DNA sequences in tissues and cells of human or animal origin. A plasmid construct encoding the PERV-pol gene or the in vitro transcribed RNA derived from the plasmid (cRNA) serves as a standard template for amplification of a 178 bp fragment. This study showed that the detection of this target sequence was linear over a range from 20 copies to 2 million copies of the plasmid and from 100 copies to 1 million copies of the cRNA. In addition, amplification of the target sequence was not inhibited by the presence of exogenous genomic DNA. These results demonstrate that a real time (TaqMan-based) PCR or RT-PCR assay can provide a sensitive, reproducible, and robust method for detecting and quantifying PERV DNA or RNA sequences in samples of human or guinea pig origin.
D000818:Animals; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D020077:Endogenous Retroviruses / Q000235:genetics / Q000302:isolation & purification*; D006168:Guinea Pigs; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013552:Swine / Q000821:virology*; D019914:Taq Polymerase
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral; D019914:Taq Polymerase