Real-Time TaqMan PCR as a Specific and More Sensitive Alternative to the Branched-Chain DNA Assay for Quantitation of Simian Immunodeficiency Virus RNA

Leutenegger, Christian M.; Higgins, Joanne; Matthews, Timothy B.; Tarantal, Alice F.; Luciw, Paul A.; Pedersen, Niels C; North, Thomas W.

doi:10.1089/088922201750063160

Cited by 109 publications

(95 citation statements)

References 42 publications

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“…The fluorogenic TaqMan system has been shown to be a fast, simple, and reliable method to quantify genomic DNA (31). Both the SRY and ⑀-globin systems have ranges of linear quantification over 5-6 log decades (21,34), and the results on the amplification efficiency and precision determination are in agreement with other TaqMan systems validated in previous studies (29,35). The specificity of the SRY assay was preserved, as we did not observe amplification signals in animals from a previous pregnancy or in gravid monkeys carrying a female fetus or genomic DNA from human male samples.…”

Section: Discussionsupporting

confidence: 80%

Quantitative Analysis of Male Fetal DNA in Maternal Serum of Gravid Rhesus Monkeys (Macaca mulatta)

Jiménez

2003

Pediatric Research

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The isolation of human fetal DNA from the maternal circulation has provided a source of fetal material for prenatal diagnosis. The objective of this study was to investigate whether a similar pattern could be observed in the maternal circulation of male-bearing gravid rhesus monkeys. A real-time PCR TaqMan system for the rhesus Y-chromosome sex determining region was used to determine fetal sex and to quantify fetal DNA concentrations. Results in 14 healthy pregnancies indicated that fetal male DNA could be routinely detected in maternal serum by 50 d of gestation (late first trimester; term 165 Ϯ 10 d). Fetal DNA concentrations increased with advancing gestation, reaching a mean of 341 genome equivalents/mL of serum (range 11-1570 copies/mL) in the last trimester of gestation, similar to findings in humans. The fetal DNA concentration corresponded to 2.7% of the total maternal serum DNA in the third trimester. Similar to findings in humans, male fetal DNA sequences were not detected postpartum (through 4 wk postpartum) or in animals with a previous history of delivering male offspring. These data indicate that fetal male DNA is present in the maternal circulation of gravid rhesus monkeys comparable to findings in humans and further support the use of this nonhuman primate species as a model to investigate fetomaternal cell trafficking and microchimerism. The discovery of high levels of fetal nucleic acids in the maternal circulation has opened up new areas of investigation and provided a potential new approach for prenatal molecular diagnosis (1). The isolation of cell-free fetal DNA has been shown to be useful material for fetal sex determination and screening for pregnancy-related complications and fetal disease (2, 3). Furthermore, this technique can potentially be applied to investigate other developmental questions associated with the level and degree of fetomaternal trafficking, maintenance of the fetal allograft and tolerance induction, and maternal autoimmune diseases such as scleroderma (4 -14). For exploring such fields of study, appropriate animal models that closely simulate human development and disease are needed.Studies in nonhuman primates, specifically macaques, have shown similarities in anatomy, developmental biology, hematology, and immunology that have prompted the use of this species as a model in fetal and pediatric research (15)(16)(17)(18)(19)(20). Monkeys and humans share many characteristic features because of their close phylogenetic relationship. Developmental similarities include spatial and temporal pattern of organ development, placental structure, and length of gestation. Unlike rodents and sheep, human and nonhuman primates (monkeys) have a similar placental structure, which highlights the importance of this model for addressing questions related to transplacental trafficking of fetal cells and DNA.In this study, we investigated the presence of male fetal rhesus DNA in maternal serum during the length of gestation. In addition, the ⑀-globin gene was used to quantify total (i.e....

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Section: Discussionsupporting

confidence: 80%

Quantitative Analysis of Male Fetal DNA in Maternal Serum of Gravid Rhesus Monkeys (Macaca mulatta)

Jiménez

2003

Pediatric Research

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“…The reaction was carried out using one tube of RT-PCR master mix (PE Applied Biosystems, CA) on the ABI Prism 7900 sequence detector (PE Applied Biosystems, CA). The data were analyzed with Sequence Detector software and quantitated using the relative computational method (15,16). TaqMan real-time PCR validation of gene expression was performed using Assays on Demand systems (Applied Biosystems, CA).…”

Section: Methodsmentioning

confidence: 99%

Rapid Onset of Intestinal Epithelial Barrier Dysfunction in Primary Human Immunodeficiency Virus Infection Is Driven by an Imbalance between Immune Response and Mucosal Repair and Regeneration

Sankaran

George

Reay

et al. 2008

J Virol

190

159

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Gut-associated lymphoid tissue (GALT) is an early target for human immunodeficiency virus type 1 (HIV-1) infection and is a site for severe CD4؉ T-cell depletion. HIV-associated enteropathy is well-documented in chronic HIV-1 infection. However, the initial host responses to HIV infection in GALT and the early molecular correlates of HIV enteropathogenesis have not been characterized during primary HIV infection. In this study, we provide evidence of viral replication in GALT resident CD4؉ T cells and macrophages in primary-stage patients and identify early patterns of host mucosal responses and changes in the molecular microenvironment through gene expression profiling. High levels of viral replication in GALT and marked CD4 ؉ T-cell depletion correlated with decreased expression levels of genes regulating epithelial barrier maintenance and digestive/ metabolic functions. These changes coincided with a marked increase in the transcription of immune activation-, inflammation-, and apoptosis-associated genes. Our findings indicate that HIV-induced pathogenesis in GALT emerges at both the molecular and cellular levels prior to seroconversion in primary HIV infection, potentially setting the stage for disease progression by impairing the ability to control viral replication and repair and regenerate intestinal mucosal tissues.

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“…Plasma viral RNA levels. A real-time quantitative RT-PCR (TaqMan) assay with a sensitivity of 50 copies of viral RNA per ml of plasma was used to quantitate RT-SHIV RNA (17).…”

Section: Methodsmentioning

confidence: 99%

Suppression of Virus Load by Highly Active Antiretroviral Therapy in Rhesus Macaques Infected with a Recombinant Simian Immunodeficiency Virus Containing Reverse Transcriptase from Human Immunodeficiency Virus Type 1

North

Rompay

Higgins

et al. 2005

J Virol

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We have modeled highly active antiretroviral therapy (HAART) for AIDS in rhesus macaques infected with a chimera (RT-SHIV) of simian immunodeficiency virus containing reverse transcriptase from human immunodeficiency virus type-1 (HIV-1). Seven RT-SHIV-infected macaques were treated with a combination of efavirenz (200 mg orally once daily), lamivudine (8 mg/kg subcutaneously once daily), and tenofovir (30 mg/kg subcutaneously once daily). Plasma viral RNA levels in all animals were reduced by more than 1,000-fold after 4 weeks and, in six of the seven animals, were reduced to undetectable levels after 10 weeks. Virus loads increased slightly between 12 and 16 weeks of treatment, associated with problems with the administration of efavirenz. After a change in the method of efavirenz administration, virus loads declined again and remained undetectable in the majority of animals for the duration of therapy. Treatment was stopped for three animals after 36 weeks of therapy, and virus loads increased rapidly. Posttreatment RT-SHIV isolates had no mutations associated with resistance to any of the three drugs. Efavirenz treatment was stopped, but lamivudine and tenofovir treatment for two other macaques was continued. The virus load in one of these two animals rebounded; virus from this animal was initially free of drug-resistance mutations but acquired the K65R mutation in reverse transcriptase at 11 weeks after efavirenz treatment was withdrawn. These results mimic HAART of HIV-1-infected humans. The RT-SHIV/rhesus macaque model should be useful for studies of tissue reservoirs and sites of residual replication that are not possible or practical with humans.

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Real-Time TaqMan PCR as a Specific and More Sensitive Alternative to the Branched-Chain DNA Assay for Quantitation of Simian Immunodeficiency Virus RNA

Cited by 109 publications

References 42 publications

Quantitative Analysis of Male Fetal DNA in Maternal Serum of Gravid Rhesus Monkeys (Macaca mulatta)

Quantitative Analysis of Male Fetal DNA in Maternal Serum of Gravid Rhesus Monkeys (Macaca mulatta)

Rapid Onset of Intestinal Epithelial Barrier Dysfunction in Primary Human Immunodeficiency Virus Infection Is Driven by an Imbalance between Immune Response and Mucosal Repair and Regeneration

Suppression of Virus Load by Highly Active Antiretroviral Therapy in Rhesus Macaques Infected with a Recombinant Simian Immunodeficiency Virus Containing Reverse Transcriptase from Human Immunodeficiency Virus Type 1

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