The reverse transcriptase (RT) assay is a simple, relatively inexpensive, w
idely used assay that can detect all retroviruses (known and novel retrovir
uses as well as infectious and defective retroviruses) on the basis of the
divalent cation requirement of their RT enzyme, i.e., Mg2+ or Mn2+. Descrip
tions of various RT assays have been published; however, they cannot be dir
ectly applied to the analysis of biological products or clinical samples wi
thout further standardization to determine the lower limit of virus detecti
on (sensitivity), assay variability (reproducibility), or ability to detect
different retroviruses (specificity). We describe the detection of type E
and type D primate retroviruses, which may be pathogenic for humans, by a n
ew P-32-based, Mg2+-containing RT assay. The results show that the sensitiv
ity of detection is <3.2 50% tissue culture infective doses (TCID(50)s) for
human immunodeficiency virus type 1 (HIV-1) and <1 TCID50 for simian immun
odeficiency virus isolated from a rhesus macaque (SIVmac). Analysis of reco
mbinant HIV-1 RT enzyme indicated that 10(-5) U, which is equivalent to 4.2
5 x 10(4) virions, could be detected. Additionally, genetically distinct ty
pe D retroviruses such as simian AIDS retrovirus and squirrel monkey retrov
irus were also detected in the assay with similar sensitivities. Thus, the
improved RT assay can be used to detect genetically divergent Mg2+-dependen
t retroviruses of human and simian origin that can infect human cells and t
hat therefore pose a potential health risk to humans.