FRugally Optimized DNA Octomer (FRODO) qPCR Measurement of HIV and SIV in Human and Nonhuman Primate Samples

Abstract

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 qPCRs. Typically, one amplicon is inserted into one plasmid construct, and the plasmid is then amplified, purified, serially diluted, and 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 normalize one to another. Here we report a single plasmid containing eight amplicons, which can be used to quantify several different strains of simian immunodeficiency virus and human immunodeficiency virus, cell number equivalents for humans and nonhuman primates, T cell receptor excision circles, and bacterial 16S DNA. This FRugally Optimized DNA Octomer (FRODO) plasmid was created and standardized to quantify all eight PCR amplicons. © 2021 US Government. Basic Protocol 1: Total genomic DNA extraction from primary cells Basic Protocol 2: Quantitative PCR for viral, bacterial, and cell number equivalents Support Protocol: Purification, quantification, and storage of FRODO standard plasmid DNA.

Keywords: HIV; SIV; nonhuman primate; qPCR.

Figure 1.

Example PCR plate layout mapping samples and FRODO standards with master mixes for each target analyte (HIV: yellow, TREC: green, 16S: red, albumin: blue)

Figure 2.

Example standard curve produced in the StepOne software for SIV in an unknown sample of cells isolated from an SIV-infected rhesus macaque.

Figure 3.

Map of FRODO plasmid indicating DNA sequences for quantification, flanking restriction enzymes, and functional plasmid components (created in Geneious Prime).

Figure 4.

Example PCR plate layout for standardizing FRODO mapping FRODO dilutions and cell lysates of approximated cell numbers as determined by cell sorter or counting. (FRODO: yellow, cell lysates: green).

Figure 5.

TaqMan qPCR amplification curves of FRODO plasmid dilutions with TREC, HIVgag, SIVmac239, CCR5, albumin, 16S, SIVsm, SIVagm, and AmpR primers and probes. Amplification curves were normalized by feature scaling reporter values from 0 to 1 for each reaction.