Figure 2. Highly Sensitive Isolation of Viral DNA from 0.4 mL of Plasma. A serial dilution of Adenovirus 5 (10⁵, 10⁴, 10³, 10² and 10¹) was spiked into 0.4 mL of plasma, and adenoviral DNA was then isolated using Norgen's Plasma/Serum Circulating DNA Purification Mini Kit (Slurry Format). Three microlitres of each 100 µL elution was then used as the template in a 20 µL PCR reaction using Ad5-specific primers, an Ad5-specific probe and Norgen's 2X PCR Master Mix (Cat #28007). The results were quantified using a standard curve that was plotted using standard adenoviral DNA of known concentration. As few as 80 pfu/mL could be isolated and detected from 0.4 mL of plasma.

Figure 3. Reproducible Recovery of Circulating DNA. Norgen's Plasma/Serum DNA Purification Mini Kit (Slurry Format) was used to isolate equal amounts of DNA fragments (300 bp) added to 20 plasma samples. DNA was purified from 0.4 mL plasma using Norgen's Plasma/Serum DNA Purification Mini Kit (Slurry Format) with an elution of 100 μL. DNA yield was quantified using real-time PCR targeting the two added DNA fragments. The blue line indicates the average percentage of recovery ± SD (91% ± 7).

Figure 4. Reproducible Recovery of Circulating DNA. Norgen's Plasma/Serum DNA Purification Mini Kit (Slurry Format) was used to isolate equal amounts of DNA fragments (1000 bp) added to 20 plasma samples. DNA was purified from 0.4 mL plasma using Norgen's Plasma/Serum DNA Purification Mini Kit (Slurry Format) with an elution of 100 µL. DNA yield was quantified using real-time PCR targeting the two added DNA fragments. The blue line indicates the average percentage of recovery ± SD (93% ± 2).

Figure 5. Detection of Human 5S Gene from 0.5 mL and 2 mL of Plasma. Norgen's Plasma/Serum Circulating DNA Purification Midi Kit (Slurry Format) was compared to a leading Competitor's kit for their ability to isolate high quality plasma DNA ready for sensitive downstream applications such as qPCR. Norgen's samples (blue) were found to amplify sooner than competitor Q's samples (red), when both 0.5 mL and 2 mL of plasma were processed, indicating a higher recovery of high quality circulating DNA present in Norgen's samples.

Figure 6. Determination of the Rate of Inhibition Present in Plasma DNA Samples. A common issue with processing high volumes of plasma is that inhibitors naturally present in plasma samples are often co-eluted, and with higher volumes of plasma comes higher concentrations of these inhibitors. Therefore, DNA was isolated from 0.5 and 2 mL of plasma using Norgen's Plasma/Serum Circulating DNA Purification Midi Kit (Slurry Format) and a leading Competitor's kit. Increasing volumes of elution (3, 6 and 9 µL) were then used in a 20 µL reaction containing 10 µL of Norgen’s 2X PCR Mastermix (Cat# 28007) spiked with SYBR green, 5 mM of either the 5S or S15 primer pair, and nuclease-free water. The PCR samples were amplified under the real-time program; 95°C for 3 minutes for an initial denaturation, 40 cycles of 95°C for 15 seconds for denaturation, 60°C for annealing and 72°C for extension. The reaction was run on an iCycler iQ Real-Time System (Bio-Rad). It was found that an increase in elution volume used in the PCR did not greatly affect the Ct value generated from Norgen samples. Competitor samples, on the other hand, were found to show a higher degree of PCR inhibition, as 9 µL of elution led to a drastic increase in Ct value. This trend was apparent for both 0.5 mL and 2 mL, as well as for 5S and S15 primers.

Figure 7. Detection of Human 5S and S15 Genes from 5 mL of Plasma. Norgen's Plasma/Serum Circulating DNA Purification Maxi Kit (Slurry Format) was compared to a leading competitor’s kit for their ability to isolate high quality plasma DNA ready for sensitive downstream applications such as qPCR. Three microliters of each elution was used in a 20 µL reaction containing 10 µL of Norgen's 2X PCR Mastermix (Cat# 28007) spiked with SYBR green, 5 mM of either the 5S or S15 primer pair, and nuclease-free water. As it can be seen, Norgen's samples (blue) were found to amplify sooner than the competitor samples (red), for both 5S and S15, indicating a higher recovery of high quality circulating DNA present in Norgen's samples.

Figure 8. Detection of Spiked Viral DNA from 5 mL of Plasma. Norgen's Plasma/Serum Circulating DNA Purification Maxi Kit (Slurry Format) was compared to a leading competitor kit for the ability to isolate viral DNA from 1 x 10⁶ spiked viral particles. Adenovirus (AdV, 1 x 10⁶) was spiked into 5mL of plasma, and total DNA was isolated from the plasma using Norgen's Plasma/Serum Circulating DNA Purification Maxi Kit (Slurry Format) or the competitor’s kit. Detection of the viral DNA was then based on the AdV DNA Binding Protein (DBP). Three microliters of each elution was used in a 20 µL reaction containing 10 µL of Norgen's 2X PCR Mastermix (Cat# 28007) spiked with SYBR green, 5 mM of the DBP primer pair, and nuclease-free water. Norgen's samples (blue) were found to amplify sooner than the competitor’s samples (red), indicating a higher recovery of viral DNA present in Norgen's samples.

Figure 9. Scalability of the Plasma/Serum Circulating DNA Purification Maxi Kit (Slurry Format). Total DNA was isolated from 0.5 mL, 5 mL and 10 mL of plasma using Norgen's Plasma/Serum Circulating DNA Purification Maxi Kit (Slurry Format) to determine the scalability of the kit. Three microliters of each elution was then used in a 20 µL reaction containing 10 µL of Norgen's 2X PCR Mastermix (Cat# 28007) spiked with SYBR green, 5 mM of the 5S primer pair, and nuclease-free water. The samples were found to amplify according to the volume processed (Figure 3A), indicating that the isolation method is robust and sensitive. When the Ct values were plotted against the volume of plasma processed (Figure 3B), the equation of the line was found to be 0.998, indicating an extremely linear relationship.

Figure 10. Determination of the Rate of Inhibition Present in Plasma DNA Samples. A common issue with processing high volumes of plasma is that inhibitors naturally present in plasma samples are often co-eluted, and with higher volumes of plasma comes higher concentrations of these inhibitors. To test for the presence of inhibitors, DNA was isolated from 5 and 10 mL of plasma using Norgen's Plasma/Serum Circulating DNA Purification Maxi Kit (Slurry Format). Increasing volumes of elution (3, 6 and 9 µL) were then used in a 20 µL PCR reaction containing 10 µL of Norgen's 2X PCR Mastermix (Cat# 28007) spiked with SYBR green, 5 mM of the 5S primer pair, and nuclease-free water. It was found that as the volume of elution used in the PCR reaction increased, the Ct values subsequently decreased in a linear fashion, indicating minimal PCR inhibition present in these plasma DNA samples.