Universal virus detection by degenerate-oligonucleotide primed polymerase chain reaction of purified viral nucleic acids.
This study describes a novel non-specific universal virus detection method that permits molecular detection of viruses in biological materials containing mixtures of cells and viruses. Samples are subjected to nuclease digestion and ultracentrifugation to separate encapsidated viral nucleic acids from cellular nucleic acids. A degenerate oligonucleotide primer PCR (DOP-PCR) that has been optimized for the non-specific amplification of virus sized genomes is then employed. Virus identification is performed by sequencing of cloned DOP-PCR products followed by sequence comparison to sequences published in GenBank. This method was used to detect a variety of DNA viruses (including HSV, VZV, SV40, AAV, and EBV) and RNA viruses (including HTLV-I, HTLV-II, influenza, and poliovirus), which were spiked into cells, constitutively expressed in cell culture, or detected in productively infected cultured cells. This novel approach was compared with a non-specific virus detection method used previously and found to be several logs more sensitive. This type of approach has potential utility in solving virus detection and discovery problems where other methods have failed.
Nanda
Santosh
S
Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda 20892-4555, United States.
Jayan
Geetha
G
Voulgaropoulou
Frosso
F
Sierra-Honigmann
Ana Maria
AM
Uhlenhaut
Christine
C
McWatters
Bernard J P
BJ
Patel
Amita
A
Krause
Philip R
PR
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004267:DNA Viruses / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity; D014709:Vero Cells; D014777:Virus Diseases / Q000175:diagnosis / Q000821:virology
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral