An RT-PCR assay for the enzyme activity of reverse transcriptase capable of detecting single virions.

We developed a sensitive assay for the enzyme activity of reverse transcriptase (RT) by setting up an RT-PCR assay containing an ample amount of exogenous RNA template but no added RT except that provided in the sample to be tested. This assay can detect less than 100 molecules of RT, which is approximately 10 times more sensitive than a conventional RT assay. The assay detects the RT activity present in single virus particles. Because the assay is general for most, if not all, RTs and is several orders of magnitude more sensitive than ELJSA or infectivity assays, it also has promise as a tool for discovery of new retroviruses. RT-PCR is conventionally used for detection of minute amounts of RNA template. In this format, exogenous RT is provided in excess to convert template RNA into amplifiable DNA. We reasoned that the assay could be 'turned around' to detect minute amounts of RT activity by setting up an RT-PCR reaction with adequate amounts of exogenous RNA template but no RT except that provided in the sample to be tested. The RNA template we used comes from Brome Mosaic Virus (BMV), an RNA virus with no DNA phase in its life cycle; this minimizes the problem of homologous DNA contaminating the template RNA. To make the assay more convenient and reduce the chance of 'carry-over' contamination, we set up the RT and PCR reactions in a single tube but in separate compartments. The reagents for the PCR reaction, minus Mg + + and template DNA, are placed in the bottom of a thin walled 'MicroAmp' PCR tube (Perkin Elmer). Above this is a layer of melted, re-congealed wax. Above the wax are the RT reactants including Mg + and sample to be assayed for RT activity. During the 37°C incubation for reverse transcription, the chance for formation of'primer-dimer' and other amplifiable artifacts is reduced because Taq polymerase is inactive in the absence of Mg + . Following reverse transcription, the heating associated with PCR melts the wax and mixes the two compartments, allowing Taq polymerase to come into contact with Mg + + and any template DNA synthesized by RT. The product of the PCR reaction is detected using a radiolabelled internal oligonucleotide which is primer extended on the PCR product in a subsequent reaction and then detected by polyacrylamide gel electrophoresis and autoradiography. The sensitivity of this assay was investigated using purified RTs and stocks of Human Immunodeficiency Virus (HTV-1) and Friend Murine Leukemia Virus (F-MuLV). The assay detects between 3 and 100 molecules of RT from HTV-1, Moloney MuLV or Avian Myeloblastosis Virus (AMV) (Figure 1, lanes 3, 6 and 9). To determine sensitivity for viral particles, we serially diluted stocks of HTV-l containing known concentrations of p24 gag protein. One virus particle is estimated to contain 0.05 fg of p24 (1). Our assay detects RT activity in samples containing as little as 0.6 to 0.03 fg of p24 (Figure 1, lanes 13 and 15). Similar results to those shown in Figure 1 were obtained with 11 other HTV-1 isolates grown in tissue culture. Standard ELJSA assays for p24 are limited in sensitivity to about 5 pg of p24. With HIV-1 or F-MuLV, our assay can detect RT activity in samples containing fewer than 10~ infectious units (Figure 1, lanes 13, 15 and 19); this implies that these virus stocks contain at least 10,000 times more physical particles than infectious particles. Our assay also detects the RTs of Human T Cell Leukemia Viruses-I and II, Simian Immunodeficiency Virus, Bovine Immunodeficiency Virus, Caprine Arthritis—Encephalitis Virus, Equine Infectious Anemia Virus and Visna Virus (not shown). While many DNA-dependent DNA polymerases, such as those from Taq, T4 and T7, are negative for RT activity when added as 'samples' in this assay, Pol I has a small amount of RT activity. Extracts from some tissue culture cells also have RT activity in this assay, although the levels are orders of magnitude lower than for high titer viral stocks. We have not yet determined whether these cell-associated RT activities reflect the products of endogenous retrovirus-like RT genes or DNA-dependent DNA polymerases. Because of its tremendous sensitivity and generality, this assay may be very useful as a tool for discovery of new retroviruses.