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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!

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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: 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%

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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: 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%

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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: 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%

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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