Primers with 5′ Flaps Improve Real-Time PCR

Afonina, Irina; Ankoudinova, Irina; Mills, Alan; Lokhov, Sergey; Huynh, Phan T.; Mahoney, Walt

doi:10.2144/000112631

Cited by 68 publications

(50 citation statements)

References 8 publications

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“…In contrast, untagged primers did not improve drastically the sensitivity of the P2-and P3-targeting assays. These results illustrate the fact that the influence of the primer tails on PCR yield is not clear, as already observed (Afonina et al, 2007).…”

Section: Discussionsupporting

confidence: 79%

Characterization of the genome of human enteroviruses: Design of generic primers for amplification and sequencing of different regions of the viral genome

Bessaud

Jégouic

Joffret

et al. 2008

Journal of Virological Methods

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This article reports the development of a simple method allowing amplification and partial sequencing of the P1, P2 and P3 genomic regions of field human enterovirus strains isolated in cell cultures, by performing PCR on cDNAs generated through a single RT reaction. A set of generic primers were designed and tested on a panel of 90 field and prototype viruses belonging to the five species of human enteroviruses. This assay was shown to amplify efficiently the targeted regions of all the 90 genomes tested. The generated amplicons were sequenced successfully without the need for gel purification.This assay could be a precious tool for laboratories interested in molecular epidemiology and evolution studies implicating a great number of human enterovirus strains isolated from human or environmental samples.

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Section: Discussionsupporting

confidence: 79%

Characterization of the genome of human enteroviruses: Design of generic primers for amplification and sequencing of different regions of the viral genome

Bessaud

Jégouic

Joffret

et al. 2008

Journal of Virological Methods

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“…The PCR protocol consisted of an initialization step at 95°C for 30 s and 50 cycles at 95°C for 5 s and 60°C for 10 s. For the detection of STs, the following primers and a fluorescent TaqMan probe were used: forward primer, 5=-GGT TAG ACC AGA TCT GAG CCT G-3= (nt 10 to 31); reverse primer, 5=-AAT AAA TCA TAA CAG TTA CGC ATG CCG AGG TC-3=; and TaqMan probe, 5=-6FAM-CTA GCT AGC CAG AGA GCT CCC AGG-BHQ1-3= (nt 28 to 51). The reverse primer was designed to bind to a universal tag sequence on the 5= end of the cDNA, and contains a short 5= AT-rich flap (underlined nucleotides) to improve qRT-PCR signals (37). The cutoff value between the ST ϩϩ and ST ϩ groups were determined according to T cell activation defined by HLA-DR ϩ /CD38 ϩ / CD8 ϩ T cells.…”

Section: Methodsmentioning

confidence: 99%

Short Intracellular HIV-1 Transcripts as Biomarkers of Residual Immune Activation in Patients on Antiretroviral Therapy

Ishizaka

Sato

Nakamura

et al. 2016

J Virol

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HIV-1 patients continue to remain at an abnormal immune status despite prolonged combination antiretroviral therapy (cART), which results in an increased risk of non-AIDS-related diseases. Given the growing recognition of the importance of understanding and controlling the residual virus in patients, additional virological markers to monitor infected cells are required. However, viral replication in circulating cells is much poorer than that in lymph nodes, which results in the absence of markers to distinguish these cells from uninfected cells in the blood. In this study, we identified prematurely terminated short HIV-1 transcripts (STs) in peripheral blood mononuclear cells (PBMCs) as an efficient intracellular biomarker to monitor viral activation and immune status in patients with cART-mediated full viral suppression in plasma. STs were detected in PBMCs obtained from both treated and untreated patients. ST levels in untreated patients generally increased with disease progression and decreased after treatment initiation. However, some patients exhibited sustained high levels of ST and low CD4؉ cell counts despite full viral suppression by treatment. The levels of STs strongly reflected chronic immune activation defined by coexpression of HLA-DR and CD38 on CD8 ؉ T cells, rather than circulating proviral load. These observations represent evidence for a relationship between viral persistence and host immune activation, which in turn results in the suboptimal increase in CD4؉ cells despite suppressive antiretroviral therapy. This cell-based measurement of viral persistence contributes to an improved understanding of the dynamics of viral persistence in cART patients and will guide therapeutic approaches targeting viral reservoirs. IMPORTANCECombination antiretroviral therapy (cART) suppresses HIV-1 load to below the detectable limit in plasma. However, the virus persists, and patients remain at an abnormal immune status, which results in an increased risk of non-AIDS-related complications. To achieve a functional cure for HIV-1 infection, activities of viral reservoirs must be quantified and monitored. However, latently infected cells are difficult to be monitored. Here, we identified prematurely terminated short HIV-1 transcripts (STs) as an efficient biomarker for monitoring viral activation and immune status in patients with cART-mediated full viral suppression in plasma. This cell-based measurement of viral persistence will contribute to our understanding of the impact of residual virus on chronic immune activation in HIV-1 patients during cART.

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“…Three qPCR assays were performed to compare the sensitivity obtained using primers designed (i) with a 5= AT-rich flap or (ii) without flaps. Two different flaps (5=-AATAAATCATAA-3= and 5=-AAAATTATTTT-3=) were used at the 5= position of the primers (21,22).…”

Section: Methodsmentioning

confidence: 99%

“…Due to its high economic impact and broad host range, P. omnivora has been included in lists of regulated organisms by the European and Mediterranean Plant Protection Organization (EPPO A1 list no. 21 [http://www.eppo.org/QUARANTINE/listA1.htm]), the California Department of Food & Agriculture (no. 3261 in the CDFA Plant Quarantine Manual, 1989 [http://pi.cdfa.ca.gov/pqm /manual/pdf/309.pdf]), and the United Nations Security Council's monitoring program of Iraq (S/1995/208 [http://www.fas.org /news/un/iraq/s/s1995-0208.htm]).…”

mentioning

confidence: 99%

Development of a Rapid, Sensitive, and Field-Deployable Razor Ex BioDetection System and Quantitative PCR Assay for Detection of Phymatotrichopsis omnivora Using Multiple Gene Targets

Arif

Fletcher

Marek

et al. 2013

Appl Environ Microbiol

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A validated, multigene-based method using real-time quantitative PCR (qPCR) and the Razor Ex BioDetection system was developed for detection of Phymatotrichopsis omnivora. This soilborne fungus causes Phymatotrichopsis root rot of cotton, alfalfa, and other dicot crops in the southwestern United States and northern Mexico, leading to significant crop losses and limiting the range of crops that can be grown in soils where the fungus is established. It is on multiple lists of regulated organisms. Because P. omnivora is difficult to isolate, accurate and sensitive culture-independent diagnostic tools are needed to confirm infections by this fungus. Specific PCR primers and probes were designed based on P. omnivora nucleotide sequences of the genes encoding rRNA internal transcribed spacers, beta-tubulin, and the second-largest subunit of RNA polymerase II (RPB2). PCR products were cloned and sequenced to confirm their identity. All primer sets allowed early detection of P. omnivora in infected but asymptomatic plants. A modified rapid DNA purification method, which facilitates a quick (ϳ30-min) on-site assay capability for P. omnivora detection, was developed. Combined use of three target genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a multigene-based, field-deployable, rapid, and reliable identification method for a fungal plant pathogen and should serve as a model for the development of field-deployable assays of other phytopathogens.

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Primers with 5′ Flaps Improve Real-Time PCR

Cited by 68 publications

References 8 publications

Characterization of the genome of human enteroviruses: Design of generic primers for amplification and sequencing of different regions of the viral genome

Characterization of the genome of human enteroviruses: Design of generic primers for amplification and sequencing of different regions of the viral genome

Short Intracellular HIV-1 Transcripts as Biomarkers of Residual Immune Activation in Patients on Antiretroviral Therapy

Development of a Rapid, Sensitive, and Field-Deployable Razor Ex BioDetection System and Quantitative PCR Assay for Detection of Phymatotrichopsis omnivora Using Multiple Gene Targets

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