Identification and evaluation of new primer sets for the detection of lentivirus proviral DNA.
We have developed sets of degenerate oligonucleotides designed to detect pol gene sequences from any member of the lentivirus subfamily when used as primers in amplification techniques such as the polymerase chain reaction (PCR). This pan-lentivirus-specific primer set (PLSPS) consists of primers, LV1, LV2, and LV3, based on conserved regions common to lentiviruses only. Our protocol is based on primary amplification with LV1 and LV2 followed by secondary amplification with a nested primer set based on the YM/VDD motif found in all reverse transcriptases (or "DDMY," in the opposite direction), and LV3, a block of lentivirus homology nested just downstream of LV1. PLSPS-PCR analysis of DNA from cells infected with HIV-1, HIV-2, SIVmac239, BIV, visna, EIAV, CAEV, OPPV, or FIV resulted in the amplification of appropriately sized products. Sequence analysis of the LV1/2 products, cloned into pBluescript (pBS), indicated that at least 20% (most often, > 80%) contained the predicted lentivirus pol sequence. Greater than 95% of the LV3/DDMY products contained the expected lentiviral sequences. Using the PLSPS, lentivirus pol sequences could typically be detected at levels of one copy in 2 x 10(6) cells after secondary amplification. No specific lentiviral PCR products were detected in DNA from uninfected human or mouse monocytes, feline or bovine leukocytes, mouse, rat or human fibroblast cell lines, chicken embryo fibroblasts, Tahr lung cells, or cell lines infected with the following retroviruses which are not lentiviruses: Rous sarcoma virus, Moloney leukemia virus or Kirsten sarcoma virus, mouse mammary tumor virus, human T-cell lymphotropic virus I, and feline leukemia virus.(ABSTRACT TRUNCATED AT 250 WORDS)
Gelman
I H
IH
Department of Microbiology, Mount Sinai Medical Center, New York, NY 10029.
Zhang
J
J
Hailman
E
E
Hanafusa
H
H
Morse
S S
SS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics*; D005069:Evaluation Studies as Topic; D015751:Genes, pol; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D004279:DNA, Viral; D015345:Oligonucleotide Probes