Ultrasensitive retrovirus detection by a reversetranscriptase assay based on product enhancement.

Abstract

Reverse transcriptase (RT) is an indispensable component of infectious retroviruses. We have developed an ultrasensitive RT test in which RNA of bacteriophage MS2 serves as the template for RT-mediated cDNA synthesis. A fragment of the cDNA is selectively amplified by polymerase chain reaction and the amplication product is analyzed by Southern blot hybridization or enzyme immunoassay. The procedure was 10'6 to 107 times more sensitive than a conventional RT test and detected as little as 10-9 unit of murine leukemia virus RT, which corresponded to 2.1 X 102 molecules, a number present in 3-11 virions. As a screening assay for filterable particle-associated RT, it was positive with supernatants from cell cultures producing human immunodeficiency virus (11V) type 1 or human T-cell leukemia virus (fITLV) type 1 or 2, but was negative with nonproducer cultures. It was positive with plasma samples from all tested individuals infected with HIV-1, HIV-2, or HTLV-1 and sera from cats infected with feline leukemia virus or feline immunodeficiency virus. Control samples from blood donors or uninfected cats were negative. Density banding experiments with culture supernatants showed that the RT activity was associated with virus particles. The assay should detect all replicationcompetent retroviruses or similar agents. It may be used as a screening assay for such agents, for quantitation of the viral load, drug susceptibility testing of RT, and control of virus inactivation in biological products.Infectious retroviruses are important causative agents of human and animal disease. They all possess a characteristic enzyme, reverse transcriptase (RT), and can thus be detected by assays for this activity (1,2). However, the current tests are insensitive when compared with virus-specific methods. Detection of human immunodeficiency virus type 1 (HIV-1) by RT assay, for example, is 100 times less sensitive than by antigen (Ag) assay (3). Polymerase chain reaction (PCR) is again orders of magnitude more sensitive (4).The narrow detection range of sequence-based tests such as PCR is, however, a disadvantage when detection of retroviruses in general is the aim. Attempts to detect uncharacterized retroviruses by the use of PCR with primers from conserved genomic regions have been met with some success but lack sensitivity and, therefore, require prior isolation and multiplication of the virus in a suitable host cell line usually difficult to find (5). Here, we report the development of an ultrasensitive RT assay capable of detecting retroviruses at a sensitivity hitherto reserved to virus-specific sequence amplification.tained from R. C. Gallo (National Institutes of Health, Bethesda, MD). HTLV-IIIB was also produced in C81-66-45 (hence called C81-66-45/HIV-1). The HTLV-1 producer MT-2 was from I. Miyoshi (Kochi Medical School, Kochi, Japan), the cloned HTLV-2 producer 76D9 was from J. Jendis (our laboratory), and the uninfected monocytoid cell line RC2A was from P. Stoeckbauer (Center Sample Pretreatment. One milliliter o...

Direct Evidence for Natural Transmission of Small-Ruminant Lentiviruses of Subtype A4 from Goats to Sheep and Vice Versa

“…All procedures relating to this work are described in detail elsewhere (11). In short, milk plasma from seropositive goats was tested for reverse transcriptase (RT) activity by the product-enhanced RT (PERT) assay (20), and 1.0 ml of the sample with the highest activity was fractionated on a 7.5 to 60% sucrose density gradient. The fraction with peak RT activity and a density of 1.15 g/ml was used for amplification of the full-length viral RNA sequence by particle-associated retroviral RNA amplification (PARRA), restricting the initial cDNA synthesis in the 5Ј rapid amplification of cDNA ends (RACE) step of PARRA to priming by primer pK1 (11).…”

Adaptation of the Fluorogenic 5′-Nuclease Chemistry to a PCR-Based Reverse Transcriptase Assay

“…Because some thermostable DNA polymerases have RNA-dependent DNA polymerase activity, background signals can be generated (10,14). Additionally, the assay is not selective for retroviruses, because lysates of all cells tested have PBRT activity (9,10,15).…”

“…The sensitivity of the RT assay was increased by using an RNA template of known sequence and PCR to amplify the cDNA product. These PCR-based RT (PBRT) assays (3,14,15) are at least a million fold more sensitive than conventional RT assays and have the capacity to detect a single retrovirus particle. These assays have also been called PERT, for product-enhanced RT, (14) and Amp-RT (3).…”

“…The legend for Figure 1 gives the sequences of the TaqMan primers and the probe designed for use. To assess the sensitivity of these new primers in comparison with the primers used previously (10,14,15), we carried out our version of the PBRT assay (10). Using (i) avian myeloblastoma virus (AMV) RT to generate the cDNA and primer extension and (ii) polyacrylamide gel electrophoresis to analyze the PCR product (10), we found that the sensitivity of the new primers with the PBRT assay was equivalent to the sensitivity of the original primers (14), which were able to detect between 100 and 1000 pU of AMV RT (data not shown).…”

“…In the PERT assay (14), the primer used for the reverse transcription step is also used as the downstream primer in the PCR amplification of the cDNA product. The legend for Figure 1 gives the sequences of the TaqMan primers and the probe designed for use.…”