Detection of feline immunodeficiency virus proviral DNA in feline peripheral blood mononuclear cells by the nested two-step polymerase chain reaction.
The polymerase chain reaction (PCR) was applied to detect feline immunodeficiency virus (FIV) proviral DNAs in primary peripheral blood mononuclear cells (PBMC). Suitable conditions for PCR amplification were examined to obtain highly sensitive and specific results by simple staining in agarose gel. Specific amplification of FIV proviral DNA in PBMC DNA of FIV-infected cats was achieved by a nested two-step PCR that amplified the DNA first with outer primers and then with inner primers nested within the first primers. PCR amplification using different primers indicated that those based on the gag sequence of the FIV/TM2 strain isolated in Japan were suitable for the detection of FIV genomes in naturally infected Japanese pet cats. By the nested two-step PCR with mixed gag primers of TM2 and Petaluma, isolated in the USA, we could detect FIV genomes in all 11 primary PBMC samples from FIV-seropositive cats tested. The PCR protocol developed here is sensitive and specific for molecular detection of FIV infection in cats.
Momoi
Y
Y
Department of Veterinary Internal Medicine, Faculty of Agriculture, University of Tokyo, Japan.
Matsumoto
Y
Y
Watari
T
T
Goitsuka
R
R
Miyazawa
T
T
Mikami
T
T
Tsujimoto
H
H
Hasegawa
A
A
D000818:Animals; D001483:Base Sequence; D002415:Cats; D017931:DNA Primers; D004279:DNA, Viral / Q000302:isolation & purification*; D016181:Feline Acquired Immunodeficiency Syndrome / Q000097:blood / Q000382:microbiology*; D016089:Immunodeficiency Virus, Feline / Q000235:genetics / Q000302:isolation & purification*; D007963:Leukocytes, Mononuclear / Q000382:microbiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D004279:DNA, Viral