Acrometrix hiv 1 panel

Manufactured by Thermo Fisher Scientific

Sourced in United States

The AcroMetrix HIV-1 panel is a laboratory product designed for the detection and quantification of HIV-1 virus. It provides a standardized reference material for use in the evaluation and validation of HIV-1 diagnostic assays.

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Lab products found in correlation

Cfx96 real time system, by Bio-Rad (4 mentions) Qiaamp viral rna mini kit, by Qiagen (4 mentions) C1000 thermal cycler, by Bio-Rad (3 mentions) Taqman fast virus 1 step master mix, by Thermo Fisher Scientific (2 mentions) Easysep human cd4 t cell enrichment kit, by STEMCELL (2 mentions) Rna ultrasense one step quantitative rt pcr system, by Thermo Fisher Scientific (1 mentions) Qiamp viral rna mini kit, by Qiagen (1 mentions) Anti cd3 cd28 dynabeads, by Thermo Fisher Scientific (1 mentions)

8 protocols using acrometrix hiv 1 panel

Linearity Assessment of HIV-1 Assay

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Linearity of HIV-1 subtype B across the assay’s dynamic range was assessed using AcroMetrix HIV-1 panel from Life Technologies (Catalog number 950470 batch number 400716). This panel has 1 HIV negative panel member and 6 HIV-1 positive panel members with concentrations ranging from 1e2 to 5e6 copies/mL. The recovered concentration of each panel member was compared to the concentration value provided by the vendor. The linearity of the various subtypes of HIV-1 was confirmed by testing 5–7 dilutions of clinical specimens (HIV subtypes A1, B, C, F1 and CRF02-AG) and cultured isolates of HIV (subtypes G and A/E and group O and N). The dilutions tested for clinical specimens targeted 1e2, 5e2, 1e3, 1e4 and 1e5 copies/mL. The concentration and available volume of the clinical sample stocks did not permit testing concentrations above 1e5 copies/mL. For the clinical isolates two additional dilutions at 1e6 copies/mL and 5e6 copies/mL were also tested.

Hatzakis A., Papachristou H., Nair S.J., Fortunko J., Foote T., Kim H., Peling T.L, & Worlock A.J. (2016). Analytical characteristics and comparative evaluation of Aptima HIV-1 Quant Dx assay with Ampliprep/COBAS TaqMan HIV-1 test v2.0. Virology Journal, 13, 176.

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Quantification of Plasma HIV-1 RNA

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Viral RNAs were extracted from plasma using QIAamp Viral RNA Mini kit (Qiagen). Plasma viral load (pVL) was determined by qRT-PCR using C1000 Thermal Cycler and CFX96 Real-Time system (Bio-Rad) with TaqMan Fast Virus 1-Step Master Mix (Life technologies), primers V1 and V2, and probe V3. The detection limit of plasma HIV-1 RNA was 200 copies/mL and was determined through repeating end point detection from serial dilution of the AcroMetrix HIV-1 Panel (Life technologies).

Yuan Z., Wang N., Kang G., Niu W., Li Q, & Guo J. (2017). Controlling multi-cycle replication of live-attenuated HIV-1 using an unnatural genetic switch. ACS synthetic biology, 6(4), 721-731.

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Analytical Performance of HIV-1 Assay

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Two standard WHO panels of HIV-1 were used to study the analytical performances of the assay. The 2010-established 3rd WHO HIV-1 international standard (National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK; NIBSC code: 10/152) is based on HIV-1 subtype B field isolate, isolated postmortem from a patient who died from AIDS-defining illness, propagated on PBMCs allowing low passage stock stored down. This 3rd WHO HIV-1 standard has been assigned a value of 185,000 international units (IU)/mL [i.e., 111,000 copies/mL, 5.04 log copies/mL, when using the conversion factor 0.58 copies/IU [12 (link)]]. The 2000-established 2nd WHO international reference panel preparation of various HIV-1 subtypes (National Institute for Biological Standards and Control; NIBSC code: 12/224) consists in a well-characterized reference panel of 10 different HIV-1 groups, including group M subtypes A, B, C, D, AE, F, G, and AA-GH, group N, and group O. The AcroMetrix® HIV-1 panel (applied Biosystems, AcroMetrix Corporation, Benicia, CA, USA) consists of HIV-1 standards at concentrations of 10,000,000, 1,000,000, 100,000, 10,000, and 1,000 IU/mL calibrated against the WHO international HIV-1 RNA standard.

Mossoro-Kpinde C.D., Mboumba Bouassa R.S., Jenabian M.A., Wolyec S.T., Robin L., Matta M., Longo J.D., Grésenguet G., Andreoletti L, & Bélec L. (2016). Analytical Performances of Human Immunodeficiency Virus Type 1 RNA-Based Amplix® Real-Time PCR Platform for HIV-1 RNA Quantification. AIDS Research and Treatment, 2016, 7954810.

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Evaluation of Amplix HIV-1 RNA Assay Accuracy

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The assay accuracy was evaluated by preparing standards at nominal concentrations of 20, 100, 1,000 (threshold of viral failure according to 2013-revised WHO criteria), 5,000, and 50,000 HIV-1 RNA copies/mL from the AcroMetrix HIV-1 panel (applied Biosystems) and quantifying HIV-1 RNA levels in each sample five times the same day by the HIV-1 RNA-based Amplix real-time PCR assay. Each sample was carried through the entire Amplix platform procedure, including specimen preparation, amplification, and detection operated by multiple users. Therefore, the estimation of the accuracy represents all aspects of the test procedure. The resulting bias (%) was calculated for each nominal standard.

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Quantifying HIV-1 RNA in Plasma

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HIV-1 RNA was extracted from plasma with QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol with on-column DNase I treatment. HIV-1 plasma viral load was quantified with RNA UltraSense One-Step Quantitative RT-PCR System (Invitrogen, Waltham, MA) using the following conditions: 50°C for 15 min; 95°C for 2 min; 40 cycles at 95°C for 15 sec and 60°C for 1 min. The primers and probe target the HIV-1 gag as previously described [17 (link)]. AcroMetrix HIV-1 panel (ThermoFisher Scientific, Fremont, CA) was used for standard curve.

Tso F.Y., Kang G., Kwon E.H., Julius P., Li Q., West J.T, & Wood C. (2018). Brain is a potential sanctuary for subtype C HIV-1 irrespective of ART treatment outcome. PLoS ONE, 13(7), e0201325.

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HIV-1 Viral Load Quantification

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Plasma viral loads were measured using qRT-PCR as described previously (89 (link)) 2 weeks after each HIV-1 rectal challenge. Briefly, a QIAamp Viral RNA mini kit (Qiagen, Cat# 52906) was used to extract viral RNA from the mouse plasma and HIV-1 analytical standards in the AcroMetrix HIV-1 panel (ThermoFisher Scientific, Cat# 950470) following the manufacturer’s protocol. Viral RNA was then quantified using a C1000 Thermal Cycler and the CFX96 Real-time system (Bio-Rad). The reaction mixture consisted of 5 µL extracted viral RNA, TaqMan Fast Virus 1-Step master mix (ThermoFisher Scientific, Cat# 4444432), forward primer (5′-GCCTCAATAAAGCTTGCCTTGA-3′), reverse primer (5’- GGGCGCCACTGCTAGAGA-3), probe (/56-FAM/CCAGAGTCA/ZEN/CACAACAGACGGGCACA/3IABkFQ/), and nuclease-free water to achieve a total volume of 20 µL.

Lohani S.C., Ramer-Tait A.E, & Li Q. (2024). High-fat diet feeding exacerbates HIV-1 rectal transmission. mSystems, 9(3), e01322-23.

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Flavopiridol Inhibits HIV Replication

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CD4+ T cells from donors with HIV were isolated from PBMCs by negative selection using the EasySepTM Human CD4+ T cell Enrichment Kit (STEM-CELL, Vancouver, BC, Canada) and cultured as described previously [56 (link)], 10⁶ cells were then treated with 0.1% DMSO or 30 nM flavopiridol for 24 hours in 1 mL cultures. Alternatively, 10⁶ cells (in 1 mL) were treated with DMSO or flavopiridol for 72 hours, washed extensively and resuspended in fresh R20+ plus IL-2 and 0.1% DMSO or 30 nM flavopiridol, incubated for an additional 72 – 96 hours, washed extensively and resuspended in fresh R20+ plus IL-2 without compounds, and incubated for a further 24 hours. Following incubation, cells were counted visually by trypan blue staining.
Viral RNA was extracted from culture supernatants using a QIAamp Viral RNA Mini kit (Qiagen, Germantown, MD, USA) and subjected to quantitative RT-PCR (qRT-PCR) as described previously using a C1000 Thermal Cycler and CFX96 Real-Time system (Bio-Rad, Hercules, CA, USA) and as described previously [59 (link)]. The limit of detection of supernatant viral RNA was 200 copies/mL as determined through repeating end point detection from serial dilution of the AcroMetrix HIV-1 Panel (Thermo Fisher, Waltham, MA, USA).

Schonhofer C., Yi J., Sciorillo A., Andrae-Marobela K., Cochrane A., Harris M., Brumme Z.L., Brockman M.A., Mounzer K., Hart C., Gyampoh K., Yuan Z., Montaner L.J, & Tietjen I. (2021). Flavonoid-based inhibition of cyclin-dependent kinase 9 without concomitant inhibition of histone deacetylases durably reinforces HIV latency. Biochemical pharmacology, 186, 114462.

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Quantifying HIV-1 Replication in CD4+ T Cells

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CD4⁺ T cells from PLWH were isolated from PBMCs by negative selection using the EasySep human CD4⁺ T-cell enrichment kit (STEM-CELL Technologies, Vancouver, BC, Canada) and cultured as described previously (19 (link)). A total of 10⁶ cells were then treated with 0.1% DMSO or test agents for 24 h in 1-mL cultures in R20+ plus 100 U/mL IL-2. Cells were then washed extensively and resuspended in fresh R20+ plus IL-2 and 0.1% DMSO in the presence of anti-CD3/CD28 Dynabeads (Invitrogen) in a 1:1 ratio for 24 h. Following incubation, live cells were counted visually by trypan blue staining. Viral RNA was extracted from culture supernatants using a QIAmp viral RNA minikit (Qiagen) and subjected to quantitative PCR, as described previously, using a C1000 thermal cycler and CFX96 real-time system (Bio-Rad) (58 (link)). The limit of detection for supernatant viral RNA was 20 copies/mL as determined through endpoint detection from serial dilution of the AcroMetrix HIV-1 panel (Thermo Fisher).

Tietjen I., Schonhofer C., Sciorillo A., Naidu M.E., Haq Z., Kannan T., Kossenkov A.V., Rivera-Ortiz J., Mounzer K., Hart C., Gyampoh K., Yuan Z., Beattie K.D., Rali T., Shuda McGuire K., Davis R.A, & Montaner L.J. (2023). The Natural Stilbenoid (–)-Hopeaphenol Inhibits HIV Transcription by Targeting Both PKC and NF-κB Signaling and Cyclin-Dependent Kinase 9. Antimicrobial Agents and Chemotherapy, 67(4), e01600-22.

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