A sensitive non-isotopic assay specific for HIV-1 associated reverse transcriptase

Porstmann, T; Meissner, K.; Gläser, Roland; Döpel, Sven-Holger; Sydow, G

doi:10.1016/0166-0934(91)90156-t

Cited by 23 publications

(14 citation statements)

References 8 publications

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“…It would be possible to use HIV-1 isolation and culture as a confirmatory test but this would be slow and expensive (15). The standard protocol applied in almost all laboratories for detection of H1V-1 with PCR consists in the amplification of clinical specimens with three primer pairs in duplicates (2,17,18).…”

Section: Resultsmentioning

confidence: 99%

Diagnosis of HIV-1 infection by PCR with two primer pairs

et al. 1993

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We have used two different primer pairs to assess HIV-1 infection of peripheral blood mononuclear cells (PBMC) by polymerase chain reaction (PCR). The study was carried out on 150 individuals: 50 seronegative individuals without risk behaviours for HIV-1 infection, 50 individuals with risk-behaviours but seronegative for HIV-1 and 50 patients with risk-behaviours who were HIV-1 seropositive. Discordances were found between the two primer pairs (SK38/39; SK68/69) in 3 cases. In the non-risk seronegative group, one specimen was scored positive with only one primer pair (SK38/SK39); all the samples belonging to seropositive individuals were found to be positive for HIV-1 DNA using both primer pairs; in the seronegative at risk group 2 samples were positive with only one primer pair (SK38/SK39), and 4 samples were found positive by both primer pairs (SK38/39 & SK68/69). Our study demonstrates that discrepant results take place with relatively high frequency; we propose that all specimens should be tested twice using at least two different primer pairs.

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Section: Resultsmentioning

confidence: 99%

Diagnosis of HIV-1 infection by PCR with two primer pairs

et al. 1993

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“…However, an isotopic assay is time-consuming and restricted to the use and disposal of radioactive material. Recently, alternative methods for measuring RT activity of HIV were developed using non-isotopically labeled nucleoside triphosphates such as BrdUTP or biotin-dUTP [8,9,11]. In Table 2 comparing PAC-RTA and the conventional non-RI RT assay kit with the same RT reaction time, PAC-RTA measured the detection limit of FIV Petaluma strain RT activity about 10 times better than the conventional kit.…”

Section: Discussionmentioning

confidence: 99%

“…However, the conventional isotopic RT assay requires laborintensive procedures and is becoming increasingly restrictive due to the use of radioactive materials. Recently, alternative methods for the RT assay using non-isotopic agents were developed [8,9,11]. We used the poly A-linked colorimetric reverse transcriptase assay (PAC-RTA) described by Suzuki et al [9] for measurement of HIV RT activity.…”

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confidence: 99%

Measurement of Reverse Transcriptase of Feline Immunodeficiency Virus by Poly A-Linked Colorimetric Assay.

Saito

Suzuki

Imai

et al. 1997

The Journal of Veterinary Medical Science

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ABSTRACT. The method of the poly A-linked colorimetric reverse transcriptase assay (PAC-RTA) was developed and evaluated for the measurement of Mg 2+ -dependent reverse transcriptase (RT) activity of feline immunodeficiency virus (FIV). PAC-RTA was first evaluated for the detection of RT activity in the culture supernatant of FIV Petaluma strain. The detection limit of RT activity by PAC-RTA was about 10-fold better than that by the conventional non-radioisotopic RT assay kit. Then, PAC-RTA was evaluated for the indication of FIV isolation from cats naturally infected with FIV. FIV was isolated from peripheral blood mononuclear cells of 9 FIV-seropositive cats. The time course appearance of RT activity measured by PAC-RTA corresponded with the analysis of FIV antigen expression by indirect immunofluorescence. Finally, PAC-RTA evaluated the drug susceptibility of FIV. MYA-1 cells (feline T-lymphoblastoid cells) were infected with FIV and were cultured in the presence of various concentrations of anti-human immunodeficiency virus agents such as azidothymidine (AZT) or dextran sulfate. An inverse relationship between the RT activities and the concentrations of these agents in the culture supernatant was confirmed by PAC-RTA. PAC-RTA is easy to perform without using radioactive materials, and one plate can handle 96 samples at one time. By monitoring the RT activity, this assay is a useful method for FIV studies such as viral replication and drug susceptibility. .25 mM MgCl 2 , 62.5 mM Tris pH7.8, 1.25 mM dithiothreitol, and 2.5 µg/ml oligo dT 12-18 (Pharmacia LKB, Uppsala, Sweden)] in microtiter wells. The reaction was carried out at 37°C for 1-15 hr and then washed 3 times with the wash buffer. After the washings, 100 µl of horseradish peroxidase conjugated streptavidin (GIBCO BRL, Gaithersburg, Maryland, U.S.A.) diluted 1:5,000 in TBSE buffer (10 mM Tris pH 7.5, 0.15 M NaCl, 1 mM EDTA and 1% BSA) was added to each well. After the plate was incubated for 20 min at 37°C, the free conjugate was removed by washing 4 times with the wash buffer. As a final step, 50 µl of 3,3',5,5'-tetramethylbenzidine (TMB; Kirkegaard and Perry Laboratories, Inc., U.S.A.) was added to each well. The plate was incubated at room temperature for 15 min. The reactions were stopped by the addition of 50 µl of 1N H 2 SO 4 . The plate was read at 450 nm with a plate reader. All assays were performed in duplicate. Virus: FIV Petaluma strain was used in this study. Crandell feline kidney (CrFK) cells persistently infected with the Petaluma strain (CrFK/Petaluma) [12] were grown

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“…The color intensity of each well is read using a standard plate reader (405 nm, reference at 620 nm) (Vassiliou et al, 2000). Several assays have been also established based on similar principles, but with variation in nucleotides modification and color developing methods (Porstmann et al, 1991;Urabe et al, 1992;Suzuki et al, 1993a, b;Odawara et al, 2002). Product-enhanced reverse transcriptase (PERT) assays were developed in order to increase the sensitivity.…”

Section: Introductionmentioning

confidence: 99%

A novel non-radioactive assay for HIV-RT (RdDp) based on pyrosequencing for high-throughput drug screening

Zhang

Sun

et al. 2010

Protein Cell

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Current in vitro assays for the activity of HIV-RT (reverse transcriptase) require radio-labeled or chemically modified nucleotides to detect reaction products. However, these assays are inherently end-point measurements and labor intensive. Here we describe a novel non-radioactive assay based on the principle of pyrosequencing coupledenzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate (PP i ), which is generated during nascent strand synthesis. The results show that our assay could monitor HIV-RT activity with high sensitivity and is suitable for rapid highthroughput drug screening targeting anti-HIV therapies due to its high speed and convenience. Moreover, this assay can be used to measure primase activity in an easy and sensitive manner, which suggests that this novel approach could be wildly used to analyze the activity of PP i -generated and ATP-free enzyme reactions.

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A sensitive non-isotopic assay specific for HIV-1 associated reverse transcriptase

Cited by 23 publications

References 8 publications

Diagnosis of HIV-1 infection by PCR with two primer pairs

Diagnosis of HIV-1 infection by PCR with two primer pairs

Measurement of Reverse Transcriptase of Feline Immunodeficiency Virus by Poly A-Linked Colorimetric Assay.

A novel non-radioactive assay for HIV-RT (RdDp) based on pyrosequencing for high-throughput drug screening

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