Quantitative assays for maedi-visna virus genetic sequences and mRNA's based on RT-PCR with real-time FRET measurements

Bjarki Gudmundsson; Helga Bjarnadottir; Steinunn Kristjansdottir; Jon J. Jonsson

doi:10.1016/S0042-6822(02)00067-3

Quantitative assays for maedi-visna virus genetic sequences and mRNA's based on RT-PCR with real-time FRET measurements

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Abstract

We developed robust, ultrasensitive, and accurate quantitative assays for maedi-visna virus (MVV) RNA and DNA genomic sequences and mRNA's expressed at various stages of lentiviral replication. Assay design was based on PCR with real-time fluorescence resonance energy transfer measurements. Specific assays were developed for gag-pol (genomic), tat, rev, env, and vif transcripts. Assay linearity ranged from 60 to 6 × 10⁷ copies of target DNA. All assays were able to detect and measure corresponding mRNA's in MVV-infected FOS cells, whereas no signal was detected in mock-treated cells. In addition, RT-PCR based on amplification of gag sequences could be used to quantify RNA genomic sequences in supernatants from infected cells. These quantitative assays can be used to study the role of genetic elements in MVV infection and pathogenesis. They also allow rapid testing of lentiviral vectors and packaging systems based on MVV.

Original language	English
Pages (from-to)	135-142
Number of pages	8
Journal	Virology
Volume	307
Issue number	1
DOIs	https://doi.org/10.1016/S0042-6822(02)00067-3
Publication status	Published - 1 Mar 2003

Bibliographical note

Funding Information: This work was supported by grants from The Icelandic Research Council Science Fund and Landspitali Science Fund. We thank Dr. Olafur S. Andresson and Dr. Valgerdur Andresdottir for the p8XSp5 plasmid and FOS cells and Dr. Katherine Staskus and Dr. Ashley Haase for the LV1-1KS2 plasmid. We thank Gudmundur H. Gunnarsson for expert assistance in preparation of the figures.

Other keywords

DNA quantification
FRET
Lentiviruses
LightCycler
Maedi-visna virus
RT-PCR
Splicing
mRNA
mRNA-quantification

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title = "Quantitative assays for maedi-visna virus genetic sequences and mRNA's based on RT-PCR with real-time FRET measurements",

abstract = "We developed robust, ultrasensitive, and accurate quantitative assays for maedi-visna virus (MVV) RNA and DNA genomic sequences and mRNA's expressed at various stages of lentiviral replication. Assay design was based on PCR with real-time fluorescence resonance energy transfer measurements. Specific assays were developed for gag-pol (genomic), tat, rev, env, and vif transcripts. Assay linearity ranged from 60 to 6 × 107 copies of target DNA. All assays were able to detect and measure corresponding mRNA's in MVV-infected FOS cells, whereas no signal was detected in mock-treated cells. In addition, RT-PCR based on amplification of gag sequences could be used to quantify RNA genomic sequences in supernatants from infected cells. These quantitative assays can be used to study the role of genetic elements in MVV infection and pathogenesis. They also allow rapid testing of lentiviral vectors and packaging systems based on MVV.",

keywords = "DNA quantification, FRET, Lentiviruses, LightCycler, Maedi-visna virus, RT-PCR, Splicing, mRNA, mRNA-quantification",

author = "Bjarki Gudmundsson and Helga Bjarnadottir and Steinunn Kristjansdottir and Jonsson, \{Jon J.\}",

note = "Funding Information: This work was supported by grants from The Icelandic Research Council Science Fund and Landspitali Science Fund. We thank Dr. Olafur S. Andresson and Dr. Valgerdur Andresdottir for the p8XSp5 plasmid and FOS cells and Dr. Katherine Staskus and Dr. Ashley Haase for the LV1-1KS2 plasmid. We thank Gudmundur H. Gunnarsson for expert assistance in preparation of the figures.",

year = "2003",

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doi = "10.1016/S0042-6822(02)00067-3",

language = "English",

volume = "307",

pages = "135--142",

journal = "Virology",

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AU - Gudmundsson, Bjarki

AU - Bjarnadottir, Helga

AU - Kristjansdottir, Steinunn

AU - Jonsson, Jon J.

N1 - Funding Information: This work was supported by grants from The Icelandic Research Council Science Fund and Landspitali Science Fund. We thank Dr. Olafur S. Andresson and Dr. Valgerdur Andresdottir for the p8XSp5 plasmid and FOS cells and Dr. Katherine Staskus and Dr. Ashley Haase for the LV1-1KS2 plasmid. We thank Gudmundur H. Gunnarsson for expert assistance in preparation of the figures.

PY - 2003/3/1

Y1 - 2003/3/1

N2 - We developed robust, ultrasensitive, and accurate quantitative assays for maedi-visna virus (MVV) RNA and DNA genomic sequences and mRNA's expressed at various stages of lentiviral replication. Assay design was based on PCR with real-time fluorescence resonance energy transfer measurements. Specific assays were developed for gag-pol (genomic), tat, rev, env, and vif transcripts. Assay linearity ranged from 60 to 6 × 107 copies of target DNA. All assays were able to detect and measure corresponding mRNA's in MVV-infected FOS cells, whereas no signal was detected in mock-treated cells. In addition, RT-PCR based on amplification of gag sequences could be used to quantify RNA genomic sequences in supernatants from infected cells. These quantitative assays can be used to study the role of genetic elements in MVV infection and pathogenesis. They also allow rapid testing of lentiviral vectors and packaging systems based on MVV.

AB - We developed robust, ultrasensitive, and accurate quantitative assays for maedi-visna virus (MVV) RNA and DNA genomic sequences and mRNA's expressed at various stages of lentiviral replication. Assay design was based on PCR with real-time fluorescence resonance energy transfer measurements. Specific assays were developed for gag-pol (genomic), tat, rev, env, and vif transcripts. Assay linearity ranged from 60 to 6 × 107 copies of target DNA. All assays were able to detect and measure corresponding mRNA's in MVV-infected FOS cells, whereas no signal was detected in mock-treated cells. In addition, RT-PCR based on amplification of gag sequences could be used to quantify RNA genomic sequences in supernatants from infected cells. These quantitative assays can be used to study the role of genetic elements in MVV infection and pathogenesis. They also allow rapid testing of lentiviral vectors and packaging systems based on MVV.

KW - DNA quantification

KW - FRET

KW - Lentiviruses

KW - LightCycler

KW - Maedi-visna virus

KW - RT-PCR

KW - Splicing

KW - mRNA

KW - mRNA-quantification

UR - https://www.scopus.com/pages/publications/0037346993

U2 - 10.1016/S0042-6822(02)00067-3

DO - 10.1016/S0042-6822(02)00067-3

M3 - Article

C2 - 12667821

SN - 0042-6822

VL - 307

SP - 135

EP - 142

JO - Virology

JF - Virology

IS - 1

ER -

Quantitative assays for maedi-visna virus genetic sequences and mRNA's based on RT-PCR with real-time FRET measurements

Abstract

Bibliographical note

Other keywords

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