Development of a reverse transcription droplet digital PCR (RT-ddPCR) assay for sensitive detection of simian immunodeficiency virus (SIV)

Abstract

Background Simian immunodeficiency virus (SIV)-infected rhesus macaques constitute an excellent model of human HIV infection. Sensitive detection of SIV RNA in cell and tissue samples from infected animals subjected to treatment regimens becomes especially critical in determining which therapeutic attempts are successful, and consequently, which interventions should be prioritized in HIV cure research. Results In this report, we describe the design and testing of a Raindance ddPCR platform-based, sensitive SIV reverse transcription droplet digital PCR (RT-ddPCR) assay by exploring the combinations of various priming conditions and reverse transcriptases, and testing one-step vs. two-step procedures, to eliminate background signal(s) and enable detection and quantification of low level target signals. Conclusions Similar reaction conditions and assay validation procedures can be explored for potential development of additional assays for other applications that require sensitive detection of low-level targets in RNA samples.

SARS-CoV-2 Subgenomic RNAs: Characterization, Utility, and Perspectives

“…Limited yields of cells or fluids from sampling procedures such as nasopharyngeal swabs, and the presence of potential inhibitors (e.g., chemical or protein contaminants) in clinical samples, require that PCR amplification and detection be highly sensitive and reliable during SARS-CoV-2 nucleic acid analysis. Digital PCR has demonstrated significant advantages in both SARS-CoV-2 gRNA [94][95][96] and sgRNA [49,97,98] studies due to its ability for absolute quantification [98][99][100][101][102][103][104][105], tolerance to inhibitors [106], increased precision at low analyte copy numbers [107][108][109], and inter-run reproducibility [110][111][112]. One additional distinct advantage of the digital PCR approach is its lower susceptibility to sequence mismatches, which is especially relevant as emerging mutations that can potentially predominate could affect the performance of real-time PCR-based assays if they occur in regions where the PCR primer and probes are located [113,114].…”

In pursuit of sensitivity: Lessons learned from viral nucleic acid detection and quantification on the Raindance ddPCR platform

“…employing a hot-start Taq polymerase). On the Raindance ddPCR platform, priming method also appears to influence assay background, as demonstrated in a recent study [61] (also see below).…”

“…indicating eradication). We previously demonstrated the significant role of reverse transcriptases (RTs) in ddPCR background signal generation [46] , [61] . One possible cause of background signals in RNA quantification is off-target priming that can occur during reaction set up and ramping up from room temperature to the annealing/polymerization temperature.…”

“…The main disadvantage associated with target/gene-specific priming is that it requires separate priming reactions for each target, which will effectively reduce each assay’s sensitivity if a limited amount of RNA needs to be divided among several reverse transcription reactions. On the Raindance ddPCR platform, gene-specific priming, when combined with a suitable reverse transcriptase, yielded clean target region background signal, distinct target positive signal clusters, and good agreement between the ddPCR reading counts with template inputs, allowing confident detection and quantification of low level, genuine signals [46] , [61] .…”

“…Raindance digital PCR system has been used to detect viruses [46] , [47] , [60] , [61] , [62] , [63] , [64] , [65] , cancer mutations or rare disease somatic mosaicism [30] , [60] , [66] , [67] , [68] , [69] , [70] , [71] , [72] , [73] , [74] , gene/DNA copy number [34] , [75] , single nucleotide polymorphism (SNP) [34] , [76] , fusion gene transcript [77] , and genetically engineered traits [57] .…”