Real time pcr

Manufactured by Abbott

Sourced in United States

Real-time PCR is a laboratory instrument used for the quantitative analysis of nucleic acid sequences. It employs the polymerase chain reaction (PCR) technique to amplify and detect specific DNA or RNA targets in real-time. The instrument precisely monitors and records the amplification of target molecules throughout the PCR process, providing quantitative data on the initial amount of the target present in the sample.

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Lab products found in correlation

Sas 9, by SAS Institute (2 mentions) Cobas ampliprep cobas taqman 48 analyser, by Roche (1 mentions) Murex hiv ag ab combination, by DiaSorin (1 mentions) Facs lysing solution, by BD (1 mentions) Cobas taqman assay, by Roche (1 mentions) Amplicor hiv 1 monitor test version 1, by Roche (1 mentions)

10 protocols using real time pcr

High-Risk HPV Genotyping: Real-Time PCR

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Genotyping was performed using two types of real-time PCR. First, Abbott™ real-time PCR was used for initial screening, with only the Abbott-positive samples retained for further HR-HPV genotype characterization using the second real-time PCR — https://maxanim.com/genetics/pcr/hpv-genotypes-14-RT-PCR-quant-ce-v67-100frt//. The Abbott™ real time PCR was performed as per the manufacturer's instructions (www.molecular.abbott/int/en/products/infectious-disease/realtime-high-risk-hpv).
Briefly, viral DNA was extracted and amplified using the Abbott real-time PCR, with simultaneous detection and genotyping of HPV 16 and HPV 18, and a pooled detection of 12 other HR-HPV genotypes (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68).
The Sacace® Biotechnologies genotyping platform HPV Genotypes 14 Real-TM Quant (www.sacace.com/manuals.htm) was used for in-vitro real-time amplification for the quantitative or qualitative detection and genotyping of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68. The analytical specificity of the HPV Genotypes 14 Real-TM Quant kit is ensured by the selection of specific primers and probes, with PCR conditions as described elsewhere (Kuassi-Kpede et al., 2021 (link)).

Tchouaket M.C., Fokam J., Sosso S.M., Semengue E.N., Yagai B., Simo R.K., Sando Z., Nka A.D., Tchinda G.P., Takou D., Fainguem N., Chenwi C., Ka'e A.C., Abba A., Zam M.K., Perno C.F., Colizzi V, & Ndjolo A. (2022). High genotypic diversity of human papillomavirus among women in Cameroon: implications for vaccine effectiveness. IJID Regions, 5, 130-136.

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Plasma HIV-1 Viral Load Quantification

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Plasma HIV-1 viral load (copies/ml) were quantified using the COBAS AmpliPrep/COBAS TaqMan 48 Analyser (Roche Diagnostic, Branchburg, NJ, USA), or by real-time PCR (Abbott, Des Plaines, IL, USA), with a detection limit of 40 copies/ml of plasma.

Li Z., Lu X., Hu Z., Luo Z., Jiang W., Wu H., Gao Y., Yan J., Zhang Q., Song A., Huang X., Mou D., Su B, & Zhang T. (2017). Syphilis Infection Differentially Regulates the Phenotype and Function of γδ T Cells in HIV-1-Infected Patients Depends on the HIV-1 Disease Stage. Frontiers in Immunology, 8, 991.

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Viral Coinfection Screening Protocol

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Blood was drawn to test for viral co-infections at the time of enrollment in the study. HIV–Analysis for HIV-1 and HIV-2 antibodies was performed by enzyme linked immuno sorbent assay (ELISA) with Murex HIV Ag/Ab combination (DiaSorin, Dartfort, UK). Confirmatory testing was done with Western Blot analysis. HTLV–Sera with HTLV-1 diagnosis by ELISA (Cambridge Biotech Corp., Worcester, MA) were confirmed by Western Blot analysis. HBV–Hepatitis B core antigen (HBc) total antibodies was determined using an Anti-HBc Architect System (Abbott Laboratories. Abbott Park, Illinois, U.S.A). HCV–HCV IgG antibodies was determined using an Anti-HCV Architect System (Abbott Laboratories. Abbott Park, Illinois, U.S.A). HCV RNA was determined by RealTime PCR (Abbot Laboratories. Abbott Park, Illinois, U.S.A).

Machado P.R., Machado L.M., Shibuya M., Rego J., Johnson W.D, & Glesby M.J. (2015). Viral Co-infection and Leprosy Outcomes: A Cohort Study. PLoS Neglected Tropical Diseases, 9(8), e0003865.

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Flow Cytometric CD4+ T-cell Enumeration

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Routine blood CD4⁺ T-cell counts (cells/μL) were measured by four-color flow cytometry using the human CD45, CD3, CD4, and CD8 cell markers (BD Biosciences) in whole peripheral blood samples from each patient processed with a fluorescence activated cell sorter (FACS) lysing solution (BD Biosciences) according to the manufacturer's instructions. The plasma HIV-1 viral load (copies per milliliter of plasma) was quantified by real-time PCR (Abbott, Des Plaines, IL, USA). The sensitivity of viral RNA detection using this assay is 40 copies/mL of plasma (BD Biosciences).

Zhang Q.Y., Zhang X., Su B., Liu L.F., Yang X.D., Tang B., Xia H., Ma P., Zhang T, & Wu H. (2020). Increased early activation of CD56dimCD16dim/- natural killer cells in immunological non-responders correlates with CD4+ T-cell recovery. Chinese Medical Journal, 133(24), 2928-2939.

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Evaluating SAMBA II HIV-1 Test Accuracy

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We calculated the overall specificity and sensitivity of the SAMBA II Qual whole blood test across all tested samples and at HIV-1 RNA thresholds of detectable (>40), 200, 400, 1000, 2000, and 3000 copies/mL. To evaluate the SAMBA II Semi-Q whole blood and Semi-Q plasma tests, we calculated the percent concordance of each SAMBA test with the Abbott RealTime PCR results at SAMBA’s approved plasma HIV-1 limit of detection of 1000 copies/mL and within the range of 1000 copies/mL +/− 0.3 log.^{41 (link)} If no valid SAMBA result was obtained using a specimen, the observation was excluded from the analyses. Sensitivity, specificity, and concordance are presented with 95% confidence intervals (CI) that were calculated using the Wilson Score method.⁵² Analyses were performed using SAS 9.4 (SAS Institute, Cary, NC).

Violette L.R., Cornelius-Hudson A., Snidarich M., Niemann L.A., Assennato S.M., Ritchie A., Goel N., Chavez P.R., Ethridge S.F., Katz D.A., Lee H., Delaney K.P, & Stekler J.D. (2022). Evaluation of SAMBA II: a qualitative and semi-quantitative HIV point-of-care nucleic acid test. Journal of acquired immune deficiency syndromes (1999), 89(5), 537-545.

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HCV Patients with Bleeding Disorders: Interferon-Free Outcomes

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Clinical data of patients with chronic HCV monoinfection and inherited bleeding disorders were retrospectively collected by five German centres. Choice and duration of interferon-free antiviral regimens were ac-cording to the treating centre and within the label of the prescribed drugs (sofosbuvir/ledipasvir 400 mg/90 mg once daily fixed-dose combination; sofosbuvir 400 mg qd; simeprevir 150 mg qd; daclatasvir 60 mg qd; paritaprevir/ritonavir/ombitasvir 75 mg/50 mg/12.5 mg qd + dasabuvir 250 mg bid).
Sustained virologic response (SVR) was defined as undetectable HCV-RNA 12 weeks after the end of therapy (SVR-12). HCV-RNA was locally analyzed by one centre with the Abbott real-time PCR (Wiesbaden, Germany, lower limit of detection 12 IU/ml) and by the other centers with the Roche Cobas TaqMan assay (Mannheim, Germany, lower limit of detection 15 IU/ml).
The study was approved by the Ethics Committee of the University of Leipzig (vote number 403-15-16112015).

Wiegand J., Schiefke I., Stein K., Berg T., Kullig U, & Ende K. (2017). Interferon-free treatment of chronic hepatitis C virus infection in patients with inherited bleeding disorders. Hamostaseologie, 37(2).

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Comprehensive Biomarker Monitoring Protocol

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The biochemical indexes were monitored three times: at the baseline, after six and twelve months where the blood samples were collected in three tubes of 4 mL. The first tube was used to determine the CD4 lymphocytes count by all patients. A volume of 50 μL of peripheral blood collected was labeled for Flow Cytometry using BD CD4 multitest kits according to Manufacturer’s instructions. Samples were the acquired using BD FACS CALIBUR II Machine (BD Biosciences, Germany). CD4 T cells values were expressed as cells/μL. The second tube was used for the determination of the viral load with an Abbott Real time PCR (Abbot, USA). HIV-1 Amplification Strategy with a detection limit of 40 copies/mL. The hemoglobin and the fasting blood sugar levels were determined in the third tube using a standard method and the « ULTRA ONE TOUCH” glucometer (LifeScan, Inc.,USA) respectively. The level of glycemia higher than ≥ 110 mg/dL was identified like cardiovascular risk factors.

Ngo-Matip M.E., Pieme C.A., Azabji-Kenfack M., Moukette B.M., Korosky E., Stefanini P., Ngogang J.Y, & Mbofung C.M. (2015). Impact of daily supplementation of Spirulina platensis on the immune system of naïve HIV-1 patients in Cameroon: a 12-months single blind, randomized, multicenter trial. Nutrition Journal, 14, 70.

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HCV Virologic Response Classification

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We defined sustained virologic response (SVR) as an undetectable HCV RNA measured between 10 and 18 weeks post-treatment. We then classified each failure to achieve SVR as either (1) an undetectable HCV RNA during treatment (ie, virologic failure), with or without early treatment discontinuation; (2) a detectable HCV RNA measurement after an undetectable measurement at any time up until 12 weeks after the end of treatment (ie, relapse); or (3) death before the assessment of SVR. HCV RNA was assessed in local laboratories using either a qualitative (COBAS Ampliprep/TaqMan HCV Test, v2.0, Roche Molecular Systems) or quantitative assay (Abbott RealTime PCR, Abbott Molecular Inc.).

Rossi C., Young J., Martel-Laferrière V., Walmsley S., Cooper C., Wong A., Gill M.J, & Klein M.B. (2019). Direct-Acting Antiviral Treatment Failure Among Hepatitis C and HIV–Coinfected Patients in Clinical Care. Open Forum Infectious Diseases, 6(3), ofz055.

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Post-SVR HCV Reinfection Monitoring

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Patients were followed post-SVR with HCV RNA measurements every 6 months until reinfection or their last study visit prior to July 2016. We defined reinfection as a single detectable HCV RNA measurement post-SVR measured in local laboratories using either a qualitative assay (COBAS Ampliprep/TaqMan HCV Test, v2.0, Roche Molecular Systems) or a quantitative assay (Abbott RealTime PCR; Abbott Molecular Inc.).

Young J., Rossi C., Gill J., Walmsley S., Cooper C., Cox J., Martel-Laferriere V., Conway B., Pick N., Vachon M.L, & Klein M.B. (2017). Risk Factors for Hepatitis C Virus Reinfection After Sustained Virologic Response in Patients Coinfected With HIV. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, 64(9), 1154-1162.

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Vitamin D, T-Cells, and Viral Loads in HIV

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25(OH)-vitamin D levels were measured at the Tufts Medical Center Core Laboratory using radioimmunoassay (DiaSorin, Stillwater, Minnesota, USA). We excluded 1 participant whose value exceeded the maximum limit of detection. Vitamin D levels were analysed both continuously and categorically as deficient versus sufficient. Vitamin D deficiency was defined as a level less than 20 ng/mL [17] (link).
T-cell subsets and HIV-1 RNA levels were measured at each study visit for HIV-infected participants (Roche Molecular Systems, Amplicor HIV-1 Monitor test version 1.5; Pleasanton, CA). Serum Hepatitis C Virus (HCV) antibody was measured at the first available visit after 2006, using an enzyme immunoassay (Ortho Diagnostics; Rochester, NY), while HCV RNA level was measured from plasma taken at or within 2 years of the vitamin D sample visit using Abbott real-time PCR (Abbott Molecular, Des Plaines, Illinois). Clinical and demographic data were obtained through study visit questionnaires; comorbidities were obtained through self-report and standardized medical record review. Race, income, insurance, healthcare resource utilization, drug use, and multivitamin intake were self-reported. Fall/winter season included September through February; spring included March through June. No levels were measured during summer months.

Lambert A.A., Drummond M.B., Mehta S.H., Brown T.T., Lucas G.M., Kirk G.D, & Estrella M.M. (2014). Risk Factors for Vitamin D Deficiency among HIV-Infected and Uninfected Injection Drug Users. PLoS ONE, 9(4), e95802.

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