Improved Mg2+-based reverse transcriptase assay for detection of primate retroviruses

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.