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Ultrasensitive retrovirus detection by a reverse transcriptase assay based on product enhancement.


Pyra, H; Böni, J; Schüpbach, J (1994). Ultrasensitive retrovirus detection by a reverse transcriptase assay based on product enhancement. Proceedings of the National Academy of Sciences of the United States of America (PNAS), 91(4):1544-1548.

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 amplification product is analyzed by Southern blot hybridization or enzyme immunoassay. The procedure was 10(6) to 10(7) 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 10(2) 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 (HIV) type 1 or human T-cell leukemia virus (HTLV) 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 replication-competent 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.

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 amplification product is analyzed by Southern blot hybridization or enzyme immunoassay. The procedure was 10(6) to 10(7) 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 10(2) 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 (HIV) type 1 or human T-cell leukemia virus (HTLV) 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 replication-competent 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.

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Additional indexing

Item Type:Journal Article, refereed
Communities & Collections:04 Faculty of Medicine > Institute of Medical Virology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:15 February 1994
Deposited On:11 Feb 2008 12:28
Last Modified:05 Apr 2016 12:22
Publisher:National Academy of Sciences
ISSN:0027-8424
Publisher DOI:10.1073/pnas.91.4.1544
Related URLs:http://www.pnas.org/cgi/content/abstract/91/4/1544
PubMed ID:7509077
Permanent URL: http://doi.org/10.5167/uzh-2155

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