Quantitation of HTLV-I proviral load by a real-time PCR assay using SYBR Green: comparison of two methods for DNA isolation. 2010

Natalia Andrea Altamirano, and Carlos Rocco, and Paula Aulicino, and Luisa Sen, and Andrea Mangano
Laboratorio de Biología Celular y Retrovirus, CONICET, Hospital de Pediatría Dr. Juan P. Garrahan, Combate de los Pozos 1881, Cuidad Autónoma de Buenos Aires, Argentina.

A real-time quantitative PCR (qPCR) assay using SYBR Green was developed to determine HTLV-I proviral load (pVL) in peripheral blood mononuclear cells (PBMCs), and its performance was evaluated with samples processed as cell lysates and DNA isolated by salting out. Primers targeting the pol region were standardized against the MT2 cell line and HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the albumin gene. The sensitivity, specificity and reproducibility of the qPCR were assessed in the two methods used for DNA processing. The assay had a limit of detection of 400 HTLV-I copies/10(6) PBMCs for both methods, with a broad range of quantitation (2.6log(10) to >5log(10)), and without cross-reactivity with HTLV-II or with HIV-1. The inter- and intra-assay coefficients of variation were less than 2.4%. HTLV-I pVL quantitation in seven blood donor samples processed as either cell lysates or isolated DNA by salting out showed a strong linear correlation and no difference in the calculated pVL (Fisher's exact test, p>0.05). The assay was found to be a low cost, robust and reproducible assay for quantifying HTLV-I pVL in samples processed as cell lysates or as isolated DNA.

UI MeSH Term Description Entries
D007963 Leukocytes, Mononuclear Mature LYMPHOCYTES and MONOCYTES transported by the blood to the body's extravascular space. They are morphologically distinguishable from mature granulocytic leukocytes by their large, non-lobed nuclei and lack of coarse, heavily stained cytoplasmic granules. Mononuclear Leukocyte,Mononuclear Leukocytes,PBMC Peripheral Blood Mononuclear Cells,Peripheral Blood Human Mononuclear Cells,Peripheral Blood Mononuclear Cell,Peripheral Blood Mononuclear Cells,Leukocyte, Mononuclear
D009930 Organic Chemicals A broad class of substances containing carbon and its derivatives. Many of these chemicals will frequently contain hydrogen with or without oxygen, nitrogen, sulfur, phosphorus, and other elements. They exist in either carbon chain or carbon ring form. Organic Chemical,Chemical, Organic,Chemicals, Organic
D011533 Proviruses Duplex DNA sequences in eukaryotic chromosomes, corresponding to the genome of a virus, that are transmitted from one cell generation to the next without causing lysis of the host. Proviruses are often associated with neoplastic cell transformation and are key features of retrovirus biology. Provirus
D011804 Quinolines
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D003959 Diamines Organic chemicals which have two amino groups in an aliphatic chain. Diamine
D004279 DNA, Viral Deoxyribonucleic acid that makes up the genetic material of viruses. Viral DNA
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D015203 Reproducibility of Results The statistical reproducibility of measurements (often in a clinical context), including the testing of instrumentation or techniques to obtain reproducible results. The concept includes reproducibility of physiological measurements, which may be used to develop rules to assess probability or prognosis, or response to a stimulus; reproducibility of occurrence of a condition; and reproducibility of experimental results. Reliability and Validity,Reliability of Result,Reproducibility Of Result,Reproducibility of Finding,Validity of Result,Validity of Results,Face Validity,Reliability (Epidemiology),Reliability of Results,Reproducibility of Findings,Test-Retest Reliability,Validity (Epidemiology),Finding Reproducibilities,Finding Reproducibility,Of Result, Reproducibility,Of Results, Reproducibility,Reliabilities, Test-Retest,Reliability, Test-Retest,Result Reliabilities,Result Reliability,Result Validities,Result Validity,Result, Reproducibility Of,Results, Reproducibility Of,Test Retest Reliability,Validity and Reliability,Validity, Face

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