Journal: bioRxiv

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

doi: 10.1101/668285

Figure Lengend Snippet: (A) Direct migration of the qPCR SIV gag DNA assay in existing format onto Raindance ddPCR platform. Left: tissue DNA containing preamplified SIV DNA as template; Right: SIV DNA standard spike-in in buffer as template. (B) The effect of modifying MgCl 2 concentration on cluster separation. (C) Background issue in the SIV target detection region using existing assay’s master mix with optimized MgCl 2 concentration.

Article Snippet: In conclusion, combining two ultrasensitive ddPCR assays for SIV nucleic acids detection with the Raindance ddPCR platform can enable significantly improved nucleic acid detection sensitivity by allowing a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

Techniques: Migration, Concentration Assay

Journal: bioRxiv

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

doi: 10.1101/668285

Figure Lengend Snippet: Performance of an MGB probe-based SIV gag ddPCR assay using TaqMan genotyping mastermix on (A) SIV and CCR5 spike-in templates and (B) ovary tissue DNA from a Rhesus macaque (311-08) infected with SIVmac239.

Article Snippet: In conclusion, combining two ultrasensitive ddPCR assays for SIV nucleic acids detection with the Raindance ddPCR platform can enable significantly improved nucleic acid detection sensitivity by allowing a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

Techniques: Infection

Journal: bioRxiv

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

doi: 10.1101/668285

Figure Lengend Snippet: (A) Sample DNA input tolerance at the droplet formation step. Droplet integrity was monitored by examining a portion of the droplets in each lane as they moved through the Source instrument during dropletization. In addition, total droplet number for each input level after dropletization (retrieved from the “RainDrop Run Completion” screen) served as another indicator of sample DNA input tolerance (Supplementary table 1). DNA sample used was duodenum DNA from Rhesus macaque 313-08. (B) Estimation of the limit of detection (LoD) of the ddPCR assay based on the Digital MIQE Guidelines . According to the guidelines, when running costs preclude optimization using ddPCR, qPCR can be used to determine certain assay parameters. (C) Performance of the SIV ddPCR assay in TaqMan genotyping mastermix in qPCR format. SIV gag DNA standard was diluted with buffer diluent. The standards were assayed as described in Materials and Methods in the following replicate format: 1 million down to 100 copies input per reaction: each in triplicates; 50, 20, 10, 7 and 5 copies input per reaction: each in 10 replicates. The data were plotted and analyzed according to the routine analyses provided in the software package with the ABI 7500 SDS instrument.

Article Snippet: In conclusion, combining two ultrasensitive ddPCR assays for SIV nucleic acids detection with the Raindance ddPCR platform can enable significantly improved nucleic acid detection sensitivity by allowing a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

Techniques: Software

Journal: bioRxiv

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

doi: 10.1101/668285

Figure Lengend Snippet: SIV ddPCR DNA assay detection of low single digit level SIV DNA input. 3 (left), 2 (middle) or 0 (right, negative control) copies of SIV DNA standard were spiked in 1 million Rhesus macaque PBMC equivalent of genomic DNA background each. Measured SIV DNA spike-in amount: 5 (left), 2(middle), 0(right).

Article Snippet: In conclusion, combining two ultrasensitive ddPCR assays for SIV nucleic acids detection with the Raindance ddPCR platform can enable significantly improved nucleic acid detection sensitivity by allowing a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

Techniques: Negative Control

Journal: bioRxiv

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

doi: 10.1101/668285

Figure Lengend Snippet: (A) Sample inhibition comparison between ddPCR and qPCR. An ovarian DNA sample (Rhesus macaque 313-08) was subjected to nested (i.e. preamplified) qPCR analysis or direct RainDance (“RD” in the table header) ddPCR analysis for SIV DNA viral load. At 3.7 M cell input per reaction (at the preamp step), SIV qPCR signal is inhibited by 99%, while at 4 M cell input per direct ddPCR reaction, SIV signal is not inhibited. (B) Quantification of Rhesus macaque necropsy tissue DNA samples (from Rhesus macaque 27882) (including an uninfected negative control sample) for SIV DNA using ddPCR. (C) Comparison between ddPCR and nested qPCR analysis results for the necropsy tissue samples in (B).

Article Snippet: In conclusion, combining two ultrasensitive ddPCR assays for SIV nucleic acids detection with the Raindance ddPCR platform can enable significantly improved nucleic acid detection sensitivity by allowing a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

Techniques: Inhibition, Negative Control

Journal: bioRxiv

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

doi: 10.1101/668285

Figure Lengend Snippet: (A) Two step RT-ddPCR assay for SIV RNA detection. 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. (B) 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 SSIV as the reverse transcriptase. The cDNA was subject to nested preamplification, and qPCR step was performed as described in Materials and Methods. The discrepancy between “measured SIV RNA copies” and “Spike-in SIV RNA copies” indicates the level of inhibition under each condition. (C) 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 reverse transcription (20 ng RNA per RT reaction). No preamplification step was performed on the cDNA.

Article Snippet: In conclusion, combining two ultrasensitive ddPCR assays for SIV nucleic acids detection with the Raindance ddPCR platform can enable significantly improved nucleic acid detection sensitivity by allowing a large amount of input DNA to be analyzed per reaction, and can overcome severe RNA inhibition when combined with suitable reverse transcription enzyme(s).

Techniques: RNA Detection, Fluorescence, Inhibition, Quantitative RT-PCR, Isolation