Langner, Charlotte (NIAID)
Quantitative polymerase chain reactions (qPCRs) are commonly employed to enumerate genes of interest among particular biological samples. Insertion of PCR amplicons into plasmid DNA is a mainstay for creation of known quantities of target sequences to standardize quantitative PCRs. Typically, one amplicon is inserted into one plasmid construct, the plasmid is then amplified, purified, serially diluted, and then quantified to be used to enumerate target sequences in unknown samples. As qPCR is often used to detect multiple amplicons simultaneously, individual qPCR standards are often desired to be normalized one to another. Unlike prior methods using separate plasmid constructs for each target sequence, FRODO incorporates eight amplicons into one plasmid construct ensuring equivalent template copy numbers for all amplicons. Amplifying, purifying, diluting and quantifying one plasmid construct rather than eight individual constructs streamlines standard curve qPCR analyses, reducing reagents and simplifying normalization between amplicons.
- Clinical Detection, Monitoring of Nucleic Acid Markers of HIV and Immunological Health: FRODO may be used to efficiently quantify target sequences in unknown samples.
- FRODO is a single plasmid containing 8 amplicons which can be used to quantify several different strains of SIV and HIV, cell number equivalents for humans and nonhuman primates, T cell receptor excision circles (humans and nonhuman primates), and bacterial 16S and ampicillin resistance DNA.
- FRODO may offer improved, more affordable, highly-sensitive nucleic acid-based HIV quantification and/or diagnostic response times, enhancing patient treatment and interventions.
- FRODO can be used to quantify levels of bacterial DNA in clinical samples to determine potential sepsis.
- This technology is especially useful in translational HIV research in which human and nonhuman primate models are used to study HIV pathogenesis, informing public health responses.
- A simplified workflow for qPCR testing. Amplifying, purifying, diluting and quantifying one plasmid construct rather than multiple, individual constructs streamlines standard curve qPCR analyses, reducing reagents and simplifying normalization between amplicons.
- At present, there are a number of antibody-based clinical tools that may be used for diagnosing/detecting HIV, but there are fewer products that affordably detect/monitor nucleic acids of HIV within cells, and immunological health, and efficacy of medicaments aimed at reducing cells infected with HIV.