Product Highlights
Figure 1: Sensitive multiplex qPCR for simultaneous detection of DNA and RNA viruses 22 replicates of each extract dilution were tested in multiplex qPCR reactions using the Low LOD 1-Step RT-qPCR Mix and the limit of detection hit rate was determined as a percentage of positive amplifications. The multiplex reactions were HAV, HBV, HCV and an extraction control and HIV-1, HIV-2, Parvovirus B19 and an extraction control. Results illustrate that Low LOD 1-Step RT-qPCR Mix can be used in multiplex reactions with both RNA and DNA viral templates, making it suitable for monitoring viral load for different types of virus. Sensitive and reproducible detection of low-level viremia for both RNA viruses (HCV, HIV-1, HIV-2 and HAV) and DNA viruses (HBV and Parvovirus B19).
HCV (RNA Virus)
HIV-1 (RNA Virus)
HAV (RNA Virus)
100
100
100
75
75
75
50
50
50
25
25
25
0
0
0
150 IU/mL 75 IU/mL 37.5 IU/mL 18.75 IU/mL 9.375 IU/mL 4.68 IU/mL
500 IU/mL 250 IU/mL 125 IU/mL 62.5 IU/mL 31.3 IU/mL 15.6 IU/mL
30 IU/mL
15 IU/mL
7.5 IU/mL 3.75 IU/mL 1.9 IU/mL 0.9 IU/mL
HIV-2 (RNA Virus)
B19 (DNA Virus)
HBV (DNA Virus)
100
100
100
75
75
75
50
50
50
25
25
25
0
0
0
30 IU/mL
15 IU/mL
7.5 IU/mL 3.75 IU/mL 1.9 IU/mL 0.9 IU/mL
500 IU/mL 250 IU/mL 125 IU/mL 62.5 IU/mL 31.3 IU/mL 15.6 IU/mL
60 IU/mL
30 IU/mL
15 IU/mL
7.5 IU/mL 3.75 IU/mL 1.9 IU/mL
Figure 2: Low limit of detection (LOD) for both RNA and DNA virus 24 replicates of purified blood viral extracts were tested in singleplex qPCR reactions using the LowLOD 1-Step RT-qPCR Mix at the indicated IU/mL. The singleplex reactions were HAV, HBV, HCV, HIV-1, HIV-2, Parvovirus B19. The amplification curve results illustrate that Low LOD 1-Step RT-qPCR Mix can be used with both RNA and DNA viruses as low as 5 IU/mL.
Amplification Curves plification Curves HCV (30 IU/mL) Amplification Curves
Amplification Curves HBV (15 IU/mL)
Amplification Curves Amplification Curves HIV-1 (60 IU/mL)
Amplification Curves
23.688 23.079 23.079 23.6 8
63.075
23.688
21.688 21.079 21.079 21.6 8
21.688
57.075
19.688 19.079 19.079 19.6 8
19.688
51.075
17.688 17.079 17.079 17.6 8
17.688
45.075
15.688 15.079 15.079 15.6 8
15.688
39.075
13.688 13.079 13.079 13.6 8
13.688
33.075
11.688 11.079 11.079 1.6 8
11.688
27.075
9.688 9.079 9.079 9.6 8
9.688
21.075
7.688 7.079 7.079 7.6 8
7.688
15.075
5.688 5.079 5.079 3.688 3.079 3.079 3.6 8 1.688 1.079 1.079 1.6 8 5.6 8
5.688
3.688
9.075
1.688
3.075
2 4 6 8 10 2 4 6 8 10 12
12 12
14 14 20 14
16 16
18 18 26
20 20
22 22
24 24 24
26 26
28 28
30 30
32 32 32
34 34 34
36 36 36
38 38 38
40 40 40
42 42 42
44 44 4
2
4
6
8
10
12
14
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18
20
22
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26
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30
32
34
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38
40
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44
10 4 16
18
22
24
28
22 30 3
34
36
38
40
42
44
2
4
6
8
12
16
18
20
26
28
30
2
4
6
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10
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44
Cycles
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Amplification Curves Amplification Curves HIV-2 (5 IU/mL)
Amplification Curves HAV (5 IU/mL) Amplification Curves
B19 (60 IU/mL)
Amplification Curves
Amplification Curves
6.628
40.432
43.077
36.932
6.028
39.077
33.432
5.428
35.077
29.932
4.828
31.077
26.432
4.228
27.077
22.932
3.628
23.077
19.432
3.028
19.077
15.932
2.428
15.077
12.432
1.828
11.077
8.932
1.228
7.077
5.432
0.628
3.077
1.932
0.028
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
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44
Cycles
Cycles
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Cycles
Cycles
Cycles
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