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Rick Conrad, Sarah A. Read, Sharon A. Matheny
Thermo Fisher Animal Health R&D
Benchmarking of qPCR and qRT-PCR master mixes Resistance to common inhibitors in animal matrix extracts
2
Background
• The Animal Health group at Thermo Fisher Scientific markets 3 qPCR master
mixes (mmx) for animal health molecular detection applications:
• Path-ID™ qPCR – for DNA targets
• AgPath-ID™ One-Step RT-PCR – for RNA targets
• Path-ID™ Multiplex One-Step RT-PCR – for multiple RNA targets in the same pot
• The master mix market is a highly competitive landscape, and we routinely test
performance against competitors.
• One relevant aspect of performance, especially for animal-related samples, is
resistance to carryover inhibitors in nucleic acid extracts.
• This study was undertaken to compare Thermo Fisher and competitor RNA and
DNA master mix performance in response to carry-over inhibitors from common
Animal Health samples.
3
Background
The following are some of our more popular tests and their test matrices :
Pathogen Species DNA/RNA Sample matrix
PRRSV Porcine RNA Oral Fluids
SIV Porcine RNA Nasal Swabs BTV Bovine RNA Whole Blood
BVDV Bovine RNA Ear Notch
Supernatant MAP Bovine DNA Feces
This study uses extracts of
each of these matrices as a
source to test inhibition of
multiple PCR master mixes.
4
• Master mix performance was evaluated by detection of a dilution series
of synthetic positive controls
• For qPCR (DNA), the assay and positive control were VetMax MAP
• For qRT-PCR (RNA), the assays and positive controls were VetMax PRRSV • This contains 2 assays, one each for NA & EU PRRSV strains
• The two lowest template concentrations were run in quadruplicate, the two
medium concentrations were run in triplicate, the highest concentration was
run in duplicate.
Study Design: Target Concentration Series
This will be combined with 8 μL of water or each sample’s eluate (“Extract”)
to generate a 25 μL reaction volume.
Master Mix
+ +
Assay
~1 copy per final reaction
~10 copies per final reaction
~100 copies per final reaction
~1,000 copies per final reaction
~10,000 copies per final reaction
• Enzyme(s)
• Buffer
• Salts
• Nucleotides
• Other additives • Proteins
• Detergents
• ????
For each assay:
• Forward primer
• Reverse primer
• TaqMan™ probe
5
• Multiple animal samples of type (“matrix”) selected were treated
• Extracted with MagMax sample prep kit
• Samples were tested to ensure no native MAP of PRRSV had been present
• For each matrix, 12 or more sample extracts were pooled
• 8 μL of each extract pool was used in each test reaction
Study Approach: Inhibitory Extracts
Single sample matrix Prepare extracts
(1 sample/animal)
Pooled
extract
8 μL of pool
added per
reaction
(~⅓ total vol)
6
Study Approach: Summary
Std curve of target ~100-104 copies/rxn
of + control
Master Mix
+ + Assay
Feces
Ear Notch Soak
Blood
Oral Fluids
water
Nasal Swabs
Pooled extract
Pooled extract
Pooled extract
Pooled extract
Pooled extract
7
Comparisons of qPCR Master Mixes
DNA Results in Graphical Format
• Each set of columns represents one master mix
• 5 columns in each set representing 5 different inputs
• 0.9, 9, 90, 900, 9000 copies per reaction
• 9 sets:
• mmx5 = PathID qPCR
• mmx1−mmx4, mmx6−mmx9 = competitors’ master mixes
• “FAM” refers to the MAP assay, which uses a FAM probe
8
DNA Master Mixes: Water (control)
24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
32.0
33.0
34.0
35.0
36.0
37.0
38.0
39.0
40.0
mmx1 mmx2 mmx3 mmx4 mmx5 mmx6 mmx7 mmx8 mmx9
av
g C
t
0.9c/rxn FAM
9c/rxn FAM
90c/rxn FAM
900c/rxn FAM
9000c/rxn FAM
PathID qPCR
140% 103% 107% 92% 98% 100% 98% 108% 120% Eff %:
9
DNA Master Mixes: Bovine Feces Extract
24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
32.0
33.0
34.0
35.0
36.0
37.0
38.0
39.0
40.0
mmx1 mmx2 mmx3 mmx4 mmx5 mmx6 mmx7 mmx8 mmx9
av
g C
t
0.9c/rxn FAM
9c/rxn FAM
90c/rxn FAM
900c/rxn FAM
9000c/rxn FAM
PathID qPCR
Eff %: 109% 115% 88% 98% 102% 79% 99% 118% 122%
10
24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
32.0
33.0
34.0
35.0
36.0
37.0
38.0
39.0
40.0
mmx1 mmx2 mmx3 mmx4 mmx5 mmx6 mmx7 mmx8 mmx9
av
g C
t
0.9c/rxn FAM
9c/rxn FAM
90c/rxn FAM
900c/rxn FAM
9000c/rxn FAM
DNA Master Mixes: Bovine Ear Notch Extract
PathID qPCR
Eff %: 111% 119% 99% 110% 106% 108% 80% 100% 100%
11
DNA Master Mixes: Bovine Blood Extract
24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
32.0
33.0
34.0
35.0
36.0
37.0
38.0
39.0
40.0
mmx1 mmx2 mmx3 mmx4 mmx5 mmx6 mmx7 mmx8 mmx9
av
g C
t
0.9c/rxn FAM
9c/rxn FAM
90c/rxn FAM
900c/rxn FAM
9000c/rxn FAM
PathID qPCR
Eff %: 97% 106% 106% 110% 105% 111% 111% 142% 102%
12
DNA Master Mixes: Porcine Oral Fluid Extract
24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
32.0
33.0
34.0
35.0
36.0
37.0
38.0
39.0
40.0
mmx1 mmx2 mmx3 mmx4 mmx5 mmx6 mmx7 mmx8 mmx9
av
g C
t
0.9c/rxn FAM
9c/rxn FAM
90c/rxn FAM
900c/rxn FAM
9000c/rxn FAM
PathID qPCR
Eff %: 109% 99% 97% 104% 106% 111% 105% 122% 100%
13
24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
32.0
33.0
34.0
35.0
36.0
37.0
38.0
39.0
40.0
mmx1 mmx2 mmx3 mmx4 mmx5 mmx6 mmx7 mmx8 mmx9
av
g C
t
0.9c/rxn FAM
9c/rxn FAM
90c/rxn FAM
900c/rxn FAM
9000c/rxn FAM
DNA Master Mixes: Porcine Nasal Swab Extract
PathID qPCR
Eff %: 171% 110% 89% 112% 100% 106% 90% 140% 94%
14
Results by Consistency Counts: DNA
DNA
qPCR (DNA)
Master Mix H2O Blood Ear Feces Oral Nasal
mmx 1 + o + + o +
mmx 2 + + + + + +
mmx 3 + + + + + +
mmx 4 + + + + + +
PathID qPCR
(mmx 5) + + + + + +
mmx 6 o o o o o o
mmx 7 o + o o + +
mmx 8 o o o + + o
mmx 9 o o o o o o
Key
Excellent
+
Detects at least 7 of 8
replicates between 0.9-9
copies/rxn. No Ct cutoff.
Good
o Detects 100% of three
replicates at 90 copies/rxn.
Poor
X
Detects 0% of three
replicates at the ‘Good’
concentration.
15
Comparisons of RT-qPCR
RNA Results in Graphical Format
• Each set of columns represents one master mix
• 5 pairs in each set representing 5 different inputs
• 4.7, 15, 47, 150, 1500 copies per reaction
• Each pair (solid & hatched) represents a different assay
• Solid = FAM channel = North American PRRSV target
• Hatched = VIC channel = European PRRSV target
• 10 sets:
• mmx2 = AgPath One-Step RT-PCR
• mmx3 = PathID Multiplex One-Step RT-PCR
• mmx1, mmx5−mmx11 = competitors’ master mixes
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24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
32.0
33.0
34.0
35.0
36.0
37.0
38.0
39.0
40.0
mmx1 mmx2 mmx3 mmx5 mmx6 mmx7 mmx8 mmx9 mmx10 mmx11
av
g C
t
4.7c/rxn FAM
4.7c/rxn VIC
15c/rxn FAM
15c/rxn VIC
47c/rxn FAM
47c/rxn VIC
150c/rxn FAM
150c/rxn VIC
1500c/rxn FAM
1500c/rxn VIC
RNA Master Mixes: Water (control)
FAM = N. American PRRS; VIC = European PRRS
PathID Multiplex One-Step RT-PCR
AgPath One-Step RT-PCR
NA 112% 93% 107% 88% 83% 106% 103% 95% 74% 113%
EU 92% 101% 105% 108% 83% 104% 94% 101% 85% 56% %eff
17
RNA Master Mixes: Bovine Feces Extract
24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
32.0
33.0
34.0
35.0
36.0
37.0
38.0
39.0
40.0
mmx1 mmx2 mmx3 mmx5 mmx6 mmx7 mmx8 mmx9 mmx10 mmx11
av
g C
t
4.7c/rxn FAM
4.7c/rxn VIC
15c/rxn FAM
15c/rxn VIC
47c/rxn FAM
47c/rxn VIC
150c/rxn FAM
150c/rxn VIC
1500c/rxn FAM
1500c/rxn VIC
PathID Multiplex One-Step RT-PCR
AgPath One-Step RT-PCR
%eff NA 84% 66% 94% 50% 29% 33% 92% 40% 67% 78%
EU 94% 50% 93% 55% 21% 19% 55% 41% 71% #DIV/0!
18
24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
32.0
33.0
34.0
35.0
36.0
37.0
38.0
39.0
40.0
mmx1 mmx2 mmx3 mmx5 mmx6 mmx7 mmx8 mmx9 mmx10 mmx11
av
g C
t
4.7c/rxn FAM
4.7c/rxn VIC
15c/rxn FAM
15c/rxn VIC
47c/rxn FAM
47c/rxn VIC
150c/rxn FAM
150c/rxn VIC
1500c/rxn FAM
1500c/rxn VIC
RNA Master Mixes: Ear Notch Soak Extract
PathID Multiplex One-Step RT-PCR
AgPath One-Step RT-PCR
%eff NA 102% 60% 94% 67% 57% 39% 65% 59% 70% 95%
EU 76% 73% 88% 59% 56% 36% 57% 63% 78% 59%
19
RNA Master Mixes: Bovine Blood Extract
24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
32.0
33.0
34.0
35.0
36.0
37.0
38.0
39.0
40.0
mmx1 mmx2 mmx3 mmx5 mmx6 mmx7 mmx8 mmx9 mmx10 mmx11
av
g C
t
4.7c/rxn FAM
4.7c/rxn VIC
15c/rxn FAM
15c/rxn VIC
47c/rxn FAM
47c/rxn VIC
150c/rxn FAM
150c/rxn VIC
1500c/rxn FAM
1500c/rxn VIC
PathID Multiplex One-Step RT-PCR
AgPath One-Step RT-PCR
%eff NA 85% 78% 94% 78% 57% 42% 108% 63% 77% 64%
EU 78% 76% 107% 76% 60% 75% 88% 84% 89% 49%
20
24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
32.0
33.0
34.0
35.0
36.0
37.0
38.0
39.0
40.0
mmx1 mmx2 mmx3 mmx5 mmx6 mmx7 mmx8 mmx9 mmx10 mmx11
av
g C
t
4.7c/rxn FAM
4.7c/rxn VIC
15c/rxn FAM
15c/rxn VIC
47c/rxn FAM
47c/rxn VIC
150c/rxn FAM
150c/rxn VIC
1500c/rxn FAM
1500c/rxn VIC
RNA Master Mixes: Porcine Oral Fluid Extract
PathID Multiplex One-Step RT-PCR
AgPath One-Step RT-PCR
%eff NA 79% 52% 79% 63% #DIV/0! #DIV/0! 65% 40% 83% 58%
EU 76% 67% 69% 53% 25% #DIV/0! 44% 40% 88% 46%
21
24.0
25.0
26.0
27.0
28.0
29.0
30.0
31.0
32.0
33.0
34.0
35.0
36.0
37.0
38.0
39.0
40.0
mmx1 mmx2 mmx3 mmx5 mmx6 mmx7 mmx8 mmx9 mmx10 mmx11
av
g C
t
4.7c/rxn FAM
4.7c/rxn VIC
15c/rxn FAM
15c/rxn VIC
47c/rxn FAM
47c/rxn VIC
150c/rxn FAM
150c/rxn VIC
1500c/rxn FAM
1500c/rxn VIC
RNA Master Mixes: Porcine Nasal Swab Extract
PathID Multiplex One-Step RT-PCR
AgPath One-Step RT-PCR
%eff NA 72% 53% 66% 49% 56% 36% 71% 36% 70% 50%
EU 90% 49% 97% 43% 53% 63% 47% 48% 70% 46%
22
Results Summary: RNA
RNA
RT-PCR Master Mix H2O Blood Ear Feces Oral Nasal
mmx 1 o o + + + AgPath One-Step RT-PCR
(mmx 2) + o x PathID Multiplex One-Step
RT-PCR (mmx 3) o +
mmx 5 + o o
mmx 6 o o x x o
mmx 7 o o x x x
mmx 8 o o o x o
mmx 9 o o x o
mmx 10 + + o
mmx 11 o o x x x
Key
Superior
Detects at least 7 of 8
replicates between 4.7-15
copies/rxn, for both assays of
the multiplex. No Ct cutoff.
Excellent
+
Detects at least 6 of 7
between 15-47 copies/rxn for
both assays of the multiplex.
Or one assay at ‘Superior’
and one at ‘Excellent’.
Good
o
Detects 100% of three
replicates at 47 copies/rxn for
both assays of the multiplex.
Or one assay at ‘Excellent’
and one at ‘Good’.
Poor
x
Detects 0% of three
replicates at 47 copies/rxn for
one or both assays of the
multiplex.
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• There is no simple conclusion
• Performance can be variable by master mix
• Different master mixes can be stronger in different matrices (different
response spectra)
• These differences can be more pronounced at low target levels
• Sometimes inhibition is only seen at very low target levels
Conclusions
© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license.
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• Sharon Matheny & Sarah Read – co-authors and actual work
• Derek Grillo – Product Manager, Animal Health
• Robert Tebbs – Sr. Staff Scientist, Animal Health
• Dan Kephart – Director of Animal Health R&D
Thank you
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