TY - JOUR
T1 - Polymerase chain reaction detection of type D simian retrovirus proviral DNA from infected macaques
AU - Pilcher, Kirsten Y.
AU - Avery, Nancy
AU - Shiigi, Stanley M.
AU - Pangares, Nancee
AU - Malley, Arthur
AU - Axthelm, Michael K.
AU - Machida, Curtis A.
N1 - Funding Information:
Authors K.Y.P. and N.A. contributed equally to this article. The authors extend special thanks to Gail Marracci for allowing us to cite her unpublished research.T he authors acknowledge the scientific advice and support of Drs. Lesley M. Hallick and Scott Wong, and the general support of current and former members of the CAM laboratory - Ms. Gail Marracci, Drs. Robert P. Searles, Julie A. Brown, and Daniel Rohrer, and Mss. Clare Midson and Valerie Nipper. The authors also acknowledge the artistic and photographics upport of Joel Ito and Vince Warren. C.A.M. and M.K.A. are supportedb y NIH NCRR 00163 and by the Medical Research Foundation of Oregon (C.A.M.). C.A.M. is a 1994 American Heart Association Established Investigator.
PY - 1994/12
Y1 - 1994/12
N2 - A simple polymerase chain reaction (PCR) approach was developed for detection of Type D simian retrovirus (SRV) serogroup 2 proviral DNA using peripheral blood lymphocytes (PBLs) obtained from infected macaques. PCR primer pairs were developed against serogroup 2 envelope (env) gene sequence, and fidelity of PCR fragment amplification was determined using molecularly cloned SRV serogroup 2 (D2/RHE/OR) DNA, and genomic DNA from Raji cells independently infected with different SRV serogroups. One primer pair exhibiting high fidelity was then utilized for PCR detection of serogroup 2 proviral DNA from PBLs, and from cells sorted into immune cell subpopulations by fluorescent-activated cell sorting (FACS). Enυ PCR fragments were readily detected from as few as 104 PBLs or immune cell subpopulations. In addition, highly specific PCR primers against serogroups 1 and 3 were utilized to detect proviral DNA from Raji cells infected with SRV serogroups. In all cases, primers designed to amplify serogroups 1,2, and 3 proviral DNA were specific for their intended serogroup. This primer information and development of a PCR approach for detection of specific SRV proviral DNA will be of potential utility as a rapid surveillance tool in monitoring type D simian retrovirus infection within Asian macaque colonies.
AB - A simple polymerase chain reaction (PCR) approach was developed for detection of Type D simian retrovirus (SRV) serogroup 2 proviral DNA using peripheral blood lymphocytes (PBLs) obtained from infected macaques. PCR primer pairs were developed against serogroup 2 envelope (env) gene sequence, and fidelity of PCR fragment amplification was determined using molecularly cloned SRV serogroup 2 (D2/RHE/OR) DNA, and genomic DNA from Raji cells independently infected with different SRV serogroups. One primer pair exhibiting high fidelity was then utilized for PCR detection of serogroup 2 proviral DNA from PBLs, and from cells sorted into immune cell subpopulations by fluorescent-activated cell sorting (FACS). Enυ PCR fragments were readily detected from as few as 104 PBLs or immune cell subpopulations. In addition, highly specific PCR primers against serogroups 1 and 3 were utilized to detect proviral DNA from Raji cells infected with SRV serogroups. In all cases, primers designed to amplify serogroups 1,2, and 3 proviral DNA were specific for their intended serogroup. This primer information and development of a PCR approach for detection of specific SRV proviral DNA will be of potential utility as a rapid surveillance tool in monitoring type D simian retrovirus infection within Asian macaque colonies.
KW - Immune cell subpopulation
KW - Peripheral blood lymphocyte
KW - Polymerase chain reaction
KW - Type D simian retrovirus
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U2 - 10.1016/0166-0934(94)90165-1
DO - 10.1016/0166-0934(94)90165-1
M3 - Article
C2 - 7714061
AN - SCOPUS:0028606546
SN - 0166-0934
VL - 50
SP - 75
EP - 86
JO - Journal of Virological Methods
JF - Journal of Virological Methods
IS - 1-3
ER -