Product-enhanced Reverse Transcriptase Assay Research Articles

Product Enhanced Reverse Transcriptase for assessing replication competent virus in vectors retroviral vectors pseudotyped with GALV and VSV-G envelopes

We evaluated the use of the Product Enhanced Reverse Transcriptase (PERT) assay as a means of detecting virus in retroviral vectors products pseudotyped with Gibbon Ape Leukemia Virus (GALV) and Vesicular Stomatitis Virus G (VSVG) envelopes. PERT provides greater standardization than the S+/L- assay which has been used extensively in virus detection. A challenge is that PERT will also detect residual retroviral vectors as vector particles contain reverse transcriptase. Vector products were cultured for 3 weeks on HEK293 cells to amplify any potential virus. In addition, vector supernatant and end-of-production cells were spiked with GALV to evaluate for inhibition by the test article. Results of PERT and the S+/L- assay were compared. PERT and S+/L- assays were both effective in detecting virus. Vector supernatants were negative at the end of 3 weeks of culture by PERT for both GAVL and VSVG pseudotyped vector. In contrast, end-of-production cells were positive by PERT due to persistent vector producing cells. A one-week culture of cell-free media obtained at the 3 weeks timepoint allowed distinction of virus-free test articles from those with virus. The PERT assay is suitable for detecting replication competent retrovirus in vector products pseudotyped with GALV and VSVG envelopes.

The duck EB66® cell substrate reveals a novel retrotransposon

During the establishment of the duck EB66® cell line as a new cell substrate for vaccine production in industry, a very low level of reverse transcriptase (RT) activity was detected in the culture supernatant by product-enhanced RT assay but a whole battery of tests failed to evidence infectious particles. Results from extensive biochemical and physical testing demonstrated that RT activity was associated to an intracellular, non-enveloped and dense structure different from an infectious retroviruses.In silico analysis of Anas platyrhynchos genome revealed that the most likely candidates for encoding a ribonucleoprotein (RNP)-associated RT were nine copies of chicken repeat 1 (CR1)-like elements, belonging to the non-long terminal repeat retrotransposons. The presence of the full length Anas platyrhynchos chicken repeat 1-like sequence (APCR1) was confirmed in EB66® cells and the related ribonucleic acid was present in the RT-containing fraction of EB66® cells.

Gammaretrovirus-specific antibodies in free-ranging and captive Namibian cheetahs.

The cheetah population in Namibia is the largest free-ranging population in the world and a key population for research regarding the health status of this species. We used serological methods and quantitative real-time PCR to test free-ranging and captive Namibian cheetahs for the presence of feline leukemia virus (FeLV), a gammaretrovirus that can be highly aggressive in populations with low genetic diversity, such as cheetahs. We also assessed the presence of antibodies to other gammaretroviruses and the responses to a FeLV vaccine developed for domestic cats. Up to 19% of the free-ranging cheetahs, 27% of the captive nonvaccinated cheetahs, and 86% of the captive vaccinated cheetahs tested positive for FeLV antibodies. FeLV-antibody-positive free-ranging cheetahs also tested positive for Rauscher murine leukemia virus antibodies. Nevertheless, FeLV was not detectable by quantitative real-time PCR and no reverse transcriptase activity was detectable by product-enhanced reverse transcriptase assay in the plasma of cheetahs or the supernatants from cultures of peripheral blood mononuclear cells. The presence of antibodies to gammaretroviruses in clinically healthy specimens may be caused either by infection with a low-pathogenic retrovirus or by the expression of endogenous retroviral sequences. The strong humoral immune responses to FeLV vaccination demonstrate that cheetahs can respond to the vaccine and that vaccination against FeLV infection may be beneficial should FeLV infection ever become a threat, as was seen in Iberian lynx and Florida panthers.

SYBR Green I-based product-enhanced reverse transcriptase assay for quantification of retroviral PFV and detection of the divalent cation preference of PFV RT

Dear Editor,Prototype foamy virus(PFV)belongs to the genus Spumavirus in the Spumaretrovirinae subfamily of Retroviridae.Although PFV and HIV have much in common,research into PFV has lagged far behind that into HIV,as PFV appeared to be non-pathogenic both in accidentally infected humans and in experimentally infected animals.In recent decades,however,more attention has been focused on PFV because it seems to be

Use of reverse-transcriptase-based HIV-1 viral load assessment to confirm low viral loads in newly diagnosed patients in Switzerland

BackgroundTreatment-naïve patients newly diagnosed with HIV occasionally present with low viral RNA of ≤1’000 copies/ml, raising concerns about viral load underestimation. Because falsely low or undetectable viral loads might lead to inadvertent virus transmission or treatment delays, confirmation of such cases by a sequence-independent viral load test is recommended in Switzerland.MethodsHIV-1 RNA ≤1’000 cp/ml by Roche’s or Abbott’s tests in patients newly diagnosed from 2010 to 2012 in Switzerland were subjected to viral load testing by the product-enhanced-reverse transcriptase (PERT) assay. These investigations were complemented with repeat and/or alternative viral RNA measurements.ResultsHIV-1 RNA ≤1’000 cp/ml was observed in 71 of 1814 newly diagnosed patients. The PERT assay suggested clinically relevant viral load underestimation in 7 of 32 cases that could be investigated. In four patients, the PERT viral load was 10-1’000-fold higher; this was confirmed by alternative HIV-1 RNA tests. Six of the 7 underestimates had been obtained with meanwhile outdated versions of Roche’s HIV-1 RNA test. In the seventh patient, follow-up revealed similar results for RNA and PERT based viral loads.ConclusionPERT assay revealed occasional severe viral load underestimation by versions of HIV-1 RNA tests meanwhile outdated. Underestimation by contemporary tests appears rare, however.

Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors

Quantification of retroviruses in cell culture supernatants and other biological preparations is required in a diverse spectrum of laboratories and applications. Methods based on antigen detection, such as p24 for HIV, or on genome detection are virus specific and sometimes suffer from a limited dynamic range of detection. In contrast, measurement of reverse transcriptase (RT) activity is a generic method which can be adapted for higher sensitivity using real-time PCR quantification (qPCR-based product-enhanced RT (PERT) assay). We present an evaluation of a modified SYBR Green I-based PERT assay (SG-PERT), using commercially available reagents such as MS2 RNA and ready-to-use qPCR mixes. This assay has a dynamic range of 7 logs, a sensitivity of 10 nU HIV-1 RT and outperforms p24 ELISA for HIV titer determination by lower inter-run variation, lower cost and higher linear range. The SG-PERT values correlate with transducing and infectious units in HIV-based viral vector and replication-competent HIV-1 preparations respectively. This assay can furthermore quantify Moloney Murine Leukemia Virus-derived vectors and can be performed on different instruments, such as Roche Lightcycler® 480 and Applied Biosystems ABI 7300. We consider this test to be an accurate, fast and relatively cheap method for retroviral quantification that is easily implemented for use in routine and research laboratories.

Detection of RD-114 virus by a reverse transcriptase assay based on product enhancement

RD-114 virus is a feline endogenous retrovirus that exists in the genome of all cats. It can be assumed that feline and canine live vaccines manufactured by culturing cells of feline origin are contaminated with the virus. The current study is attempted to develop a product enhanced reverse transcriptase (PERT) assay to detect replication-competent RD-114 virus. Since culture supernatants of Crandell-Rees feline kidney (CRFK) cells do not have detectable reverse transcriptase activity in case of do not passage on other cell lines, these results raise the possibility that RD-114 virus grow efficiently in other retrovirus producing cell lines. For the PERT assay, RD-114 virus isolated from CRFK cells may need to be passaged more than four times on 293T cells to be expressed at appreciable levels. The PERT assay described here provides an accurate method for determining the presence of RD-114 in culture supernatants and will help monitor live vaccines produced in endogenous virus producing cell lines.

Investigations on the diagnosis and retroviral aetiology of renal neoplasia in budgerigars (Melopsittacus undulatus)

The high susceptibility of budgerigars (Melopsittacus undulatus) to neoplasia, and specifically renal neoplasia, has often been reported. Further investigations led to a suspicion of a retrovirus as the causative agent for renal neoplasia in budgerigars, but definitive proof has yet to be found. In the present study, 32 budgerigars suspected of having renal neoplasia (based on the clinical presentation) were examined. The objectives were to investigate the use of different diagnostic methods for the ante-mortem diagnosis of this condition and to find more supporting evidence of a retroviral aetiology. The predominant clinical signs observed in budgerigars with renal neoplasia were lameness and absence of deep pain sensation of one leg. Alterations in haematology, plasma chemistry, and urine analyses could not pinpoint the cases of renal neoplasia. Contrast radiography of the intestinal tract proved to be diagnostically more useful compared with plain radiographic studies. Histology confirmed the renal neoplasia as adenocarcinoma. Investigations for virus identification included product-enhanced reverse transcriptase assay and enzyme-linked immunosorbent assay for the detection of avian leucosis virus group-specific antigen. Cell cultures and electron microscopy were performed on a limited number of patients. These investigations could find no presence of an exogenous, replicating retrovirus, neither could viral particles be detected by electron microscopy. Based on the current findings, it can be concluded that there is no evidence of retroviral involvement in the occurrence of renal neoplasia in budgerigars.

Evaluation of different RT enzyme standards for quantitation of retroviruses using the single-tube fluorescent product-enhanced reverse transcriptase assay

PCR-based reverse transcriptase (RT) assays are highly sensitive for broad detection of retroviruses. These assays are currently used for demonstrating the absence of retroviral contamination in vaccines and can also be applied to clinical and laboratory research to investigate low-virus replication. A single-tube fluorescent product-enhanced reverse transcriptase assay (STF-PERT) has been published that was highly sensitive for retrovirus detection (<10 virions), with enhanced reproducibility and increased efficiency [Sears, J.F., Khan, A.S., 2003. Single-tube fluorescent product-enhanced reverse transcriptase assay with AmpliWax (STF-PERT) for retrovirus quantitation. J. Virol. Meth. 108, 139–142]. In this report, the step-by-step setup and performance of the STF-PERT assay is described and sensitivity, reproducibility and specificity of the assay reported using three different RTs as standards: avian myeloblastosis virus (AMV) RT, murine leukemia virus (MMLV) RT, and human immunodeficiency virus type 1 (HIV-1) RT. Evaluation of virus stocks showed about 1–2 logs difference in RT detection and retrovirus quantitation with the different RT enzyme standards; in general, virus determination using HIV-1 RT was comparable to using the relevant virus RT.

A one-step SYBR Green I-based product-enhanced reverse transcriptase assay for the quantitation of retroviruses in cell culture supernatants

PCR-enhanced reverse transcriptase assays (PERT) are sensitive tools for the detection of retroviruses in biological samples. The adaptation of real-time PCR techniques based on fluorescent probes (F-PERT) has added a reliable quantitative capacity to the assay. In the interest of economy and time, the SYBR Green I-based real-time detection system was used to establish a convenient one-step PERT assay (SG-PERT). This assay can be completed in 2h, is linear over six orders of magnitude and can be used to quantify retroviruses belonging to divergent species, such as the human immunodeficiency virus type 1 (HIV-1), murine leukemia virus (MLV) and prototypic foamy virus (PFV).

A modified single-tube one-step product-enhanced reverse transcriptase (mSTOS-PERT) assay with heparin as DNA polymerase inhibitor for specific detection of RTase activity

Background Current regulations and recommendations proposed for the production of vaccines in continuous cell lines of any origin demand that these be free of exogenous viruses, particularly retroviruses. Recently, the ultra-sensitive product-enhanced reverse transcriptase (PERT) assay can be used to detect minute of reverse transcriptase (RTase) in single retroviral particle and is 10 6 times more sensitive than the conventional RTase assays. However, coincidental with this increase in sensitivity is an increase in false-positive reactions derived from contaminating cellular DNA polymerases, which are known to have RTase-like activities. Objectives To develop a modified single-tube one-step PERT (mSTOS-PERT) assay with improvements on decreasing significantly the level of false-positive reactions, and to evaluate the mSTOS-PERT assay for sensitivity and specificity. Study design Ampliwax™ was used to compartmentalize the reverse transcription (RT) and PCR step in the same micro-tube with more efficiency and reproducibility, while maintaining the high sensitivity. The DNA amplification products were separated by 2% agarose gel electrophoresis, and then analyzed by non-isotopic Southern blot hybridization. A wide variety of cell lines used in biologicals production were detected to validate the improved mSTOS-PERT assay. Results The detection limit for the mSTOS-PERT assay was at least 10 −9 U, when using AMV-RTase as a positive control. Furthermore, heparin involvement in the RT step can eliminate completely the false-positive PERT signals which are exhibited by cellular polymerases such as DNA-dependent DNA polymerase α, γ released by cell death. Most mammalian cells (MRC-5, Vero, WISH, 2BS, RK-13, MDCK, etc.) are PERT-negative in cell supernatants. Some PERT-positive signals in cell lysates were found to be introduced by the cellular DNA polymerases and could be inhibited specifically by heparin. Chick cells derived from either chick embryo fibroblasts (CEF) or allantoic fluid from SPF embryonated eggs, murine hybridoma cell SP2/0, etc., contained authentic RTase activities, which could not be inactivated by heparin. Conclusions The improved mSTOS-PERT assay described here may distinguish the genuine RTase activity from cellular polymerases with high sensitivity and specificity, and is rapid and easy to perform to screen for the possible contamination of minute retroviruses in the cell substrates used in vaccine production.

Product-Enhanced Reverse Transcriptase Assay for Replication-Competent Retrovirus and Lentivirus Detection

The product-enhanced reverse transcriptase (PERT) assay has been used to detect reverse transcriptase (RT) activity associated with retroviruses. Although the PERT assay has been proposed as a method for detection of replication-competent retrovirus (RCR) and lentivirus (RCL), it has not been rigorously compared with existing methods for RCR and RCL detection. We have assessed the PERT assay for detection of RCL and RCR that may contaminate lentiviral and retroviral vectors and compared it with published methods for RCL (p24gag ELISA/gag PCR) and RCR (S+/L-) detection. Our results suggest that the PERT assay is as sensitive as p24gag ELISA and gag PCR for detection of replication-competent HIV-1 in an RCL detection assay. Comparison of detection of replication-competent retroviruses, GALV and RD114, by extended S+/L- and PERT assays indicates that both assays can detect 1 IU of each virus. Our findings suggest that the PERT assay can be used for RCL and RCR testing of a variety of retroviral vectors regardless of the structure, sequence, and envelope of the vectors.

825. Product Enhanced Reverse Transcriptase (PERT) Assay for RCL/RCR Detection

Product Enhanced Reverse Transcriptase (PERT) assay has been used extensively to detect reverse transcriptase (RT) activity associated with retroviruses. We have assessed the usefulness of the PERT assay for RCL and RCR testing and compared it with existing assays for RCL (p24gag ELISA/gag PCR) and RCR (S+/L|[minus]|) detection. As a first step towards evaluating the usefulness of PERT for RCL detection, we determined the sensitivity of detection of purified and virus associated HIV-RT by PERT. The PERT assay was able to detect |[sim]| 100 molecules of purified HIV-1 RT and 1-0.1 IU of a replication competent HIV-1 virus, R7-GFP, in two independent experiments. In a RCL detection assay comprising of a 3-week amplification phase and a 1-week indicator phase, 1 IU of R7-GFP was detected by all three assays (p24 ELISA, gag PCR and PERT) in spite of higher backgrounds associated with the PERT assay. To provide additional support for the use of PERT for RCL testing, we also examined the effect of competing vector particles on RCL detection by p24 ELISA and PERT. Both assays were able to detect 1 IU of R7-GFP mixed with varying concentrations (100, 1000, 5000 and 10,000 ng of p24) of a HIV-1 vector, CS-CGW (provided by Philip Zoltick, Philadelphia, PA), in the RCL detection assay. These results suggest that the PERT assay is as sensitive as p24gag ELISA and gag PCR for detection of replication competent HIV-1 in a RCL detection assay. For evaluating the ability of the PERT assay to detect RCR, we determined the sensitivity of detection of purified M-MuLV-RT and compared the detection of replication competent retroviruses (GALV-SEATO, RD114, and 4070A) by PERT and S+/L- assays. The PERT assay detected approximately 100 molecules of purified MuLV-RT in three independent experiments. Both assays detected 1 IU of RD114 and 10 IU of 4070A; detection of the GALV-SEATO virus was more sensitive by the S+/L- assay (1-10 IU) than the PERT assay (10-100 IU). These studies indicate that the sensitivity of detection of retroviruses is equivalent by PERT and S+/L- assays. To evaluate the sensitivity of PERT in a RCR detection assay, GALV-SEATO and RD114 viruses were amplified for 3-weeks and serial dilutions of amplified material were tested by S+/L- and PERT assays. Both assays detect 1 IU of RD114 and GALV after the amplification phase suggesting that they have similar sensitivities in an extended RCR assay. In conclusion, the PERT assay can be used for RCL and RCR testing of a variety of retroviral vectors regardless of the structure, sequence, and envelope of the vectors.

82. Adaptation of a Lentiviral Vector Producer Cell Line to Reduced-Serum, Suspension Culture for Large Scale Production

The ability of lentiviral-based vectors to integrate into non-dividing cells such as neuronal cells has been exploited to correct a number of disease models. Using vectors based on the equine infectious anaemia virus (EIAV) we have previously reported the correction of an animal model of Parkinson's disease (Azzouz et al. J Neurosci. 2002 Dec 1;22(23):10302–12). To use such vectors in the clinic each manufactured lot must be tested and demonstrated to be free of replication competent lentivirus (RCL). We have designed the vectors in such a way to limit the possibility of homologous recombination taking place; the vector genome contains only 824 nt of EIAV sequence with no coding regions; all the accessory genes have been removed entirely from the system; the gag/pol sequence has been codon optimised and a heterologous envelope is used (VSV-G). However, there is still the formal possibility of some form of recombination leading to the generation of an RCL. We have therefore developed an RCL assay based on the property common to all members of the Retroviradae family, namely the presence of reverse transcriptase (RT). RT activity can be detected by a sensitive assay: Product Enhanced Reverse Transcriptase (PERT). The test article is passaged on human cells to amplify any replicating entity and then a PERT assay is carried out on the supernatant. A typical set of assay results will be presented.

Single-tube fluorescent product-enhanced reverse transcriptase assay with Ampliwax™ (STF-PERT) for retrovirus quantitation

A TaqMan fluorescent probe-based product enhanced reverse transcriptase (RT) assay is described in which the RT and polymerase chain reaction (PCR) steps are set-up in a single tube, in two compartments separated by Ampliwax™ (designated as single-tube fluorescent product-enhanced reverse transcriptase assay (STF-PERT)). This simplification of the two-step method resulted in increased assay reproducibility and handling efficiency while maintaining the sensitivity of the PERT assay (<10 virions). The STF-PERT assay can be used to quantitate low amounts of retrovirus in clinical and research materials and to evaluate retrovirus contamination in cell substrates and biological products in human use.

Evaluation of a Quantitative Product-enhanced Reverse Transcriptase Assay to Monitor Retrovirus in mAb Cell-culture

Murine hybridoma cells used in the production of monoclonal antibodies (mAbs) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAbs intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by evaluation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In a previous report, we demonstrated the utility of TaqMan fluorogenic 5′-nuclease product-enhanced reverse transcriptase (TM-PERT) assays for measuring reverse transcriptase (RT) activity in laboratory-scale cell-culture samples and RT removal by laboratory-scale models of processing steps. In this report, we evaluate the specificity, accuracy, range, precision and robustness of TM-PERT for this purpose. We find that this assay detects RT activity contained in xenotropic murine leukemia virus (X-MuLV) and CHO cell type C particles and quantifies particle numbers comparably to other assays (e.g. transmission electron microscopy, viral sequence specific TaqMan). Cell derived DNA polymerases appear to contribute only modestly to the assay background and RT activity in clarified cell culture harvests is contained largely in Type C particles. TM-PERT is linear and precise between 107and 1013pU/ml, establishing the assay range. The assay is robust in that test article storage condition and DNA/protein content had little impact on assay performance. Thus, TM-PERT appears to be an acceptable assay to measure type C particles in rodent cell culture samples.

Early detection of endogenous retroviruses in chemically induced mouse cells

Endogenous retroviral sequences are present as an integral part of eukaryotic genomes. Although the majority of these sequences are defective, a few can produce infectious virus, either spontaneously upon long-term culture or by treatment with various chemical or other agents. Early, extensive studies of retrovirus induction were done in mouse cells; however, similar studies have not been done using state-of-the-art virus detection assays and with cells of other mammalian species. To investigate induction and detection of occult retroviruses in cells of different species, especially primate cells that are used in production of biologics, we have initially determined the optimum conditions for retrovirus induction in chemically treated K-BALB mouse cells using highly sensitive product-enhanced reverse transcriptase (PERT) assays as well as transmission electron microscopy (TEM). Retrovirus induction was detected at day 1 post-drug treatment under all test conditions but was optimum using 30 μg ml −1 of 5-iododeoxyuridine (IdU) for 24 h. Additionally, the combination of IdU and 5-azacytidine specifically enhanced activation of type C particles. RT activity was detected by PERT assays in one microliter equivalent of test sample and retroviral particle production was seen by TEM analysis. The induction of infectious murine leukemia retroviruses was confirmed by infectivity assays and correlated with PERT activity. These results indicate that strategies for detection of occult viral agents should include optimization of induction conditions using multiple viral detection assays to evaluate virus activation.

Use of a quantitative product-enhanced reverse transcriptase assay to monitor retrovirus levels in mAb cell-culture and downstream processing.

Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAb's intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In this report, we assess the utility of the TaqMan fluorogenic 5'-nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assay for measuring reverse transcriptase (RT) activity in cell-culture samples and RT removal by models of processing steps. The levels of RT activity contained in laboratory-scale cell-culture harvests (10(8)-10(13) pU/mL) were substantially above the detection limit of the TM-PERT assay ( approximately 10(6) pU/mL). The nature of the RT activity from cell culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight viral particles. In laboratory-scale chromatographic runs, sufficient RT activity was present in mAb-containing eluates to accurately calculate its log(10) reduction value (LRV), typically between 2 and 4 log(10) per step. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicate that the TM-PERT assay, because it is quantitative and highly sensitive and can be used to analyze a large number of samples in a short period, is ideally suited to investigate and optimize retrovirus clearance in purification processes.

Detection of reverse transcriptase activity in the serum of patients with motor neurone disease

The recognition that both human and murine retroviruses can cause motor neurone disease-like syndromes has raised the possibility that a retrovirus may be involved in the aetiology of motor neurone disease. This possibility was explored by looking for evidence of reverse transcriptase in the serum of motor neurone disease patients. Sera from 56 patients with motor neurone disease and 58 controls were tested by the product-enhanced reverse transcriptase assay, a technique that is approximately a million fold more sensitive than conventional reverse transcriptase assays and capable of detecting very low numbers of retroviral particles. Cell-free reverse transcriptase activity was detected in the serum of 33 of the 56 motor neurone disease patients (59%) but in only 3 of the controls (P < 0.00001). The reverse transcriptase activity was detectable in the presence of a large excess of an effective inhibitor of human cellular DNA polymerases and was therefore tentatively considered to be compatible with a retroviral origin. The reverse transcriptase activity, however, was not found to be due to the presence of known human exogenous retroviruses including HIV-1, HIV-2, HTLV-I, HTLV-II, HRV-5 or human foamy virus, as assessed by PCR-based assays. Further investigations will be required to determine the source of the reverse transcriptase activity observed in these motor neurone disease patient sera.

One-Step Fluorescent Probe Product-Enhanced Reverse Transcriptase Assay

Recently developed PCR-based reverse transcriptase (RT) assays are useful in the detection of retroviruses since they are approximately a millionfold more sensitive than conventional RT assays. However, these assays are both labor- and time-intensive. The previously described product-enhanced reverse transcriptase (PERT) assay involves a two-step RT-PCR followed by detection and quantitation of PCR products by either Southern blot or enzyme-linked immunosorbent assay (ELISA). We have modified the PERT assay to be a one-step, fluorescent probe, PCR-based RT assay that can be completed from sample dilution to final quantitative assay results in approximately 5 h without loss of assay sensitivity or specificity. The assay has a dynamic range of 6 logs, and therefore, extensive sample dilution is not necessary for quantitation. This newly enhanced fluorescent PERT assay can play an important role in the high-throughput detection of retroviral infection and characterization of RT activity.