Study 2:
Aim: The performance of Air-Dryable™ Direct RNA/DNA qPCR Saliva (MDX131) was compared to Air-Dryable™ 1-Step RT-qPCR Mix (MDX095) in both air-dried and liquid format, for the detection of Flu B in saliva samples. Method: Influenza B (Flu B) samples were either processed by bead RNA extraction (sample IFB-1), or left as a crude lysate and treated with 100 µL of storage buffer #1 (sample IFB-2), storage buffer #2 (sample IFB-3), lysis buffer #1 (sample IFB-4) or lysis buffer #4. The Flu B samples were then briefly spun to collect the supernatant and human saliva from healthy individuals was added to dilute the samples by 10 3 or 10 4 (refer to Table 1). Samples were tested using standard cycling conditions (55°C x 10 min (RT step), 95°C x 3 min, followed by 5 cycles at 95°C x 10 sec and 60°C for 60 sec, and 40 cycles of 95°C x 10 sec and 60°C x 30 sec) or fast cycling conditions (55°C x 5 min, 95°C x 1 min, followed by 5 cycles 95°C x 1 sec and 60°C x 15 sec, and 38 cycles of 95°C x 1 sec and 60°C x 15 sec).
Patient Sample
Sample preparation
1 IFB-1-1 Extracted RNA from Flu B diluted 10 3 1 IFB-1-2 Extracted RNA from Flu B diluted 10 4
2 IFB-2-1 10 3 diluted Flu B in human saliva, treated with storage buffer #1 2 IFB-2-2 10 4 diluted Flu B in human saliva, treated with storage buffer #1 3 IFB-3-1 10 3 diluted Flu B in human saliva, treated with storage buffer #2 3 IFB-3-2 10 4 diluted Flu B in human saliva, treated with storage buffer #2 4 IFB-4-1 10 3 diluted Flu B in human saliva Diluted Flu B in human saliva, treated with lysis buffer #1 4 IFB-4-2 10 4 diluted Flu B in human saliva treated with lysis buffer #1 5 IFB-5-1 10 3 diluted Flu B in human saliva, treated with lysis buffer #2 5 IFB-5-2 10 4 diluted Flu B in human saliva, treated with lysis buffer #2 NC DEPC water Table 1 . Samples used for Flu B detection in human saliva samples using Air-Dryable ™ Direct RNA/DNA qPCR Saliva (MDX131) and Air-Dryable 1-Step RT-qPCR (MDX095) mixes. Samples were spiked with Influenza B viruses 1-5 as described in the table notes.
A. Standard Cycle
40
35
30
25
20
15
10
5
0
IFB-1-1 IFB-1-2
IFB-2-2 IFB-3-1 IFB-3-2 IFB-5-1 IFB-5-2 IFB-NC IFB-NC IFB-2-1
Sample
B. Fast Cycle
40
35
30
25
20
15
10
5
0
IFB-1-1 IFB-1-2
IFB-2-2 IFB-3-1 IFB-3-2 IFB-5-1 IFB-5-2 IFB-NC IFB-NC IFB-2-1
Sample
Air Dried (MDX095) | Wet (MDX095) | Air Dried (MDX131) | Wet (MDX131) | Reference Mix
Figure 2. Detection of Flu B from human saliva RNA-extracted and crude lysate samples using the Air-Dryable™ Direct RNA/DNA qPCR Saliva (MDX131) and Air-Dryable™ 1-Step RT-qPCR (MDX095) Mixes and either (A) standard or (B) fast cycling conditions. Air-dried MDX131 was the most efficient at detecting Flu B, followed by wet MDX131, then wet MDX095, dry MDX095 and the reference mix in both the fast and standard cycles.
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