Journal: PLoS ONE

Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

doi: 10.1371/journal.pone.0233085

Figure Lengend Snippet: (A) Two step RT-ddPCR reverse transcription step condition tests. In these tests, SIV RNA standard was spiked in 1 ug cellular RNA isolated from naïve Rhesus macaque PBMC. Various conditions such as different reverse transcriptases, RT enzyme amounts, and gene-specific (GSP) vs. random hexamer (HEX) priming methods were tested. The SIV RNA measured copies after ddPCR were compared to input copies to identify optimal RT conditions. Blue: SIV RNA input (copies); Orange: SIV RNA measured (copies). M-MLV: Moloney Murine Leukemia Virus Reverse Transcriptase; SSIII: SuperScript III Reverse Transcriptase. (B) Two step RT-ddPCR assay for SIV RNA detection. Different copy numbers of SIV RNA standard or buffer control were spiked in 1ug total RNA from naïve animal PBMC, and the samples were subject to reverse transcription (SIV gene-specific priming, RT enzyme: M-MLV, 200U/reaction), dropletization, end-point PCR and fluorescence reading/counting. (C) Linear dynamic range of the SIV RT-ddPCR RNA assay derived from quantification data of Fig 7B. (D) Severe RNA inhibition in a bone marrow aspirate sample (from Rhesus macaque 29676) in RT-qPCR analysis. Different amount of RNA sample extracted from a bone marrow aspirate sample, treated or untreated through a G-50 column as indicated, then subject to reverse transcription either using M-MLV or SuperScript IV (SSIV) as the reverse transcriptase. The cDNA was subject to nested preamplification, and qPCR step was performed as described in Methods. The discrepancy between “measured SIV RNA copies” and “Spike-in SIV RNA copies” indicates the level of inhibition under each condition. (E) LiCl treatment of RNA sample removes heparin inhibition in RT-qPCR analysis but RNA recovery is low. RNA samples from 4 bone marrow needle aspirate samples (from Rhesus macaque 28808, 29211, 28885 and 29341) were subject to additional precipitation step(s) (isopropanol, lithium chloride, or a sequential combination of both) before SIV RNA quantitation analysis by adding 1000 copies of SIV RNA standard. (F) SSIV RT combined with SIV ddPCR assay overcomes bone marrow RNA sample inhibition. RNA was isolated from the bone marrow aspirate, and subject to SSIV or SSIII reverse transcription (20 ng RNA per RT reaction) dropletization, end-point PCR and fluorescence reading/counting. No preamplification step was performed on the cDNA.

Article Snippet: We report the development, validation and optimization of a RainDance ddPCR DNA assay, and a RainDance reverse transcription (RT)-ddPCR RNA assay for simian immunodeficiency virus (SIV), which is widely used in nonhuman primate models for HIV/AIDS studies.

Techniques: Isolation, Random Hexamer Labeling, RNA Detection, Fluorescence, Derivative Assay, Inhibition, Quantitative RT-PCR, Quantitation Assay