PubMed · 8650330
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B Kawakami, M Takahashi, H Inoue, Y Kawamura. 1996. [Quantitative PCR].. https://pubmed.ncbi.nlm.nih.gov/8650330/
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Plant viruses pose a significant threat to global agriculture and require efficient tools for their timely detection. We present AutoPVPrimer, an innovative pipeline that integrates artificial intelligence (AI) and machine learning to accelerate the development of plant virus primers. The pipeline uses Biopython to automatically retrieve different genomic sequences from the NCBI database to increase the robustness of the subsequent primer design. The design_primers_with_tuning module uses a random forest classifier that optimizes parameters and provides flexibility for different experimental conditions. Quality control measures, including the evaluation of poly-X content and melting temperature, increase primer reliability. Unique to AutoPVPrimer is the visualize_primer_dimer module, which supports the visual evaluation of primer dimers-a feature missing in other tools. Primer specificity is validated via primer BLAST, which contributes to the overall efficiency of the pipeline. AutoPVPrimer has been successfully applied to the tomato mosaic virus, proving its adaptability and efficiency. The modular design allows customization by the user and extends the applicability to different plant viruses and experimental scenarios. The pipeline represents a significant advance in primer design and provides researchers with an effective tool to accelerate molecular biology experiments. Future developments aim to extend compatibility and incorporate user feedback to consolidate AutoPVPrimer as an innovative contribution to the bioinformatics toolbox and a promising resource for the advancement of plant virology research.
We have developed a quantitative real-time PCR assay for HTLV-I DNA. This assay approach uses real-time monitoring of fluorescent signal generation as a consequence of Taq-mediated amplification of specific target sequences to allow real-time kinetic analysis of amplicon production. This kinetic approach yields excellent sensitivity and an extremely broad linear dynamic range, and ensures that quantitation is based on analysis during the exponential phase of amplification, regardless of the input template copy number. The HTLV-I DNA assay has a nominal threshold sensitivity of 10 copy Eq/reaction, although single-copy plasmid template can be detected at frequencies consistent with statistical prediction. The linear dynamic range is in excess of 5 logs. Interassay reproducibility averages 14% (coefficient of variation) for control templates over a range of 10(1) to 10(6) copy Eq/reaction and 25%, based on studies of extraction and analysis of replicate aliquots of PBMC specimens from HTLV-I-infected subjects. The primer/probe combination targets tax sequences conserved across described HTLV-I and HTLV-II isolates. Parallel quantitation in the same samples of an endogenous sequence present at a known copy number per cell allows normalization of results for potential variation in DNA recovery. Availability of this assay should facilitate studies of basic pathogenesis and clinical evaluation of HTLV-I and HTLV-II infection, as well as assessment of therapeutic approaches.
The effect of a male steroid hormone, 5DHT, on the expression of TNF-alpha was examined using a human leukemia T cell line, Jurkat. Cells were treated with 5DHT in the presence or absence of PHA, and RNA was isolated followed by a reverse transcriptase - mediated PCR (RT-PCR) to measure the steady state levels of TNF-alpha mRNA. The treatment of cells with 5DHT resulted in a 50% of decrease in the level of TNF-alpha mRNA compared to that in untreated conditions (basal level). A similar level of reduction of the message by 5DHT was also observed in PHA-stimulated cells. The reduction of the steady state levels of TNF-alpha mRNA in Jurkat cells was a result of destabilization of the gene as demonstrated by actinomycin D treatment; a half-life of TNF-alpha message in 5DHT treated cells and non-treated cells was 1 hr and 2.5 hr, respectively, whereas that in 5DHT/PHA and PHA-treated cells was 3hr and 6hr, respectively.