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Identification of TL-Om1, an adult T-cell leukemia (ATL) cell line, as reference material for quantitative PCR for human T-lymphotropic virus 1.

ABSTRACT: Quantitative PCR (qPCR) for human T-lymphotropic virus 1 (HTLV-1) is useful for measuring the amount of integrated HTLV-1 proviral DNA in peripheral blood mononuclear cells. Many laboratories in Japan have developed different HTLV-1 qPCR methods. However, when six independent laboratories analyzed the proviral load of the same samples, there was a 5-fold difference in their results. To standardize HTLV-1 qPCR, preparation of a well-defined reference material is needed. We analyzed the integrated HTLV-1 genome and the internal control (IC) genes of TL-Om1, a cell line derived from adult T-cell leukemia, to confirm its suitability as a reference material for HTLV-1 qPCR. Fluorescent in situ hybridization (FISH) showed that HTLV-1 provirus was monoclonally integrated in chromosome 1 at the site of 1p13 in the TL-Om1 genome. HTLV-1 proviral genome was not transferred from TL-Om1 to an uninfected T-cell line, suggesting that the HTLV-1 proviral copy number in TL-Om1 cells is stable. To determine the copy number of HTLV-1 provirus and IC genes in TL-Om1 cells, we used FISH, digital PCR, and qPCR. HTLV-1 copy numbers obtained by these three methods were similar, suggesting that their results were accurate. Also, the ratio of the copy number of HTLV-1 provirus to one of the IC genes, RNase P, was consistent for all three methods. These findings indicate that TL-Om1 cells are an appropriate reference material for HTLV-1 qPCR.

SUBMITTER: Kuramitsu M

PROVIDER: S-EPMC4298509 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Identification of TL-Om1, an adult T-cell leukemia (ATL) cell line, as reference material for quantitative PCR for human T-lymphotropic virus 1.

Kuramitsu Madoka M Okuma Kazu K Yamagishi Makoto M Yamochi Tadanori T Firouzi Sanaz S Momose Haruka H Mizukami Takuo T Takizawa Kazuya K Araki Kumiko K Sugamura Kazuo K Yamaguchi Kazunari K Watanabe Toshiki T Hamaguchi Isao I

Journal of clinical microbiology 20141210 2

Quantitative PCR (qPCR) for human T-lymphotropic virus 1 (HTLV-1) is useful for measuring the amount of integrated HTLV-1 proviral DNA in peripheral blood mononuclear cells. Many laboratories in Japan have developed different HTLV-1 qPCR methods. However, when six independent laboratories analyzed the proviral load of the same samples, there was a 5-fold difference in their results. To standardize HTLV-1 qPCR, preparation of a well-defined reference material is needed. We analyzed the integrated ...[more]

PMID: 25502533

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Project description:The human rhinovirus (RV) is a positive-stranded RNA virus that causes respiratory tract diseases affecting both the upper and lower halves of the respiratory system. RV enhances its replication by concentrating RNA synthesis within a modified host membrane in an intracellular compartment. RV infections often occur alongside infections caused by other respiratory viruses, and the RV virus may remain asymptomatic for extended periods. Alongside qualitative detection, it is essential to accurately quantify RV RNA from clinical samples to explore the relationships between RV viral load, infections caused by the virus, and the resulting symptoms observed in patients. A reference material (RM) is required for quality evaluation, the performance evaluation of molecular diagnostic products, and evaluation of antiviral agents in the laboratory. The preparation process for the RM involves creating an RV RNA mixture by combining RV viral RNA with RNA storage solution and matrix. The resulting RV RNA mixture is scaled up to a volume of 25 mL, then dispensed at 100 µL per vial and stored at -80 °C. The process of measuring the stability and homogeneity of RV RMs was conducted by employing reverse transcription droplet digital polymerase chain reaction (RT-ddPCR). Digital PCR is useful for the analysis of standards and can help to improve measurement compatibility: it represents the equivalence of a series of outcomes for reference materials and samples being analyzed when a few measurement procedures are employed, enabling objective comparisons between quantitative findings obtained through various experiments. The number of copies value represents a measured result of approximately 1.6 × 105 copies/μL. The RM has about an 11% bottle-to-bottle homogeneity and shows stable results for 1 week at temperatures of 4 °C and -20 °C and for 12 months at a temperature of -80 °C. The developed RM can enhance the dependability of RV molecular tests by providing a precise reference value for the absolute copy number of a viral target gene. Additionally, it can serve as a reference for diverse studies.

Dataset Information

Identification of TL-Om1, an adult T-cell leukemia (ATL) cell line, as reference material for quantitative PCR for human T-lymphotropic virus 1.

Publications

Identification of TL-Om1, an adult T-cell leukemia (ATL) cell line, as reference material for quantitative PCR for human T-lymphotropic virus 1.

Similar Datasets

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