Benchmarking of qpcr and qrt-pcr master mixes Resistance to common inhibitors in animal matrix extracts
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1 Benchmarking of qpcr and qrt-pcr master mixes Resistance to common inhibitors in animal matrix extracts Rick Conrad, Sarah A. Read, Sharon A. Matheny Thermo Fisher Animal Health R&D The world leader in serving science
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. 2
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. 3
4 Study Design: Target Concentration Series 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. Enzyme(s) Buffer Salts Nucleotides Other additives Proteins Detergents???? Master Mix For each assay: Forward primer Reverse primer TaqMan probe + + 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 This will be combined with 8 μl of water or each sample s eluate ( Extract ) to generate a 25 μl reaction volume. 4
5 Study Approach: Inhibitory Extracts 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 Pooled extract 8 μl of pool added per reaction (~⅓ total vol) Single sample matrix Prepare extracts (1 sample/animal) 5
6 Study Approach: Summary water Oral Fluids Master Mix + + Assay Std curve of target ~ copies/rxn of + control Pooled extract Pooled extract Pooled extract Pooled extract Nasal Swabs Blood Ear Notch Soak Pooled extract Feces 6
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 7
8 DNA Master Mixes: Water (control) avg Ct Eff %: % 103% 107% 92% 98% 100% 98% 108% 120% c/rxn FAM c/rxn FAM c/rxn FAM c/rxn FAM c/rxn FAM mmx1 mmx2 mmx3 mmx4 mmx5 PathID mmx6 mmx7 mmx8 mmx9 qpcr 8
9 avg Ct DNA Master Mixes: Bovine Feces Extract Eff %: 109% 115% 88% 98% 102% 79% 99% 118% 122% c/rxn FAM c/rxn FAM c/rxn FAM c/rxn FAM c/rxn FAM mmx1 mmx2 mmx3 mmx4 mmx5 mmx6 mmx7 mmx8 mmx9 PathID qpcr 9
10 DNA Master Mixes: Bovine Ear Notch Extract avg Ct Eff %: 111% 119% 99% 110% 106% 108% 80% 100% 100% c/rxn FAM c/rxn FAM c/rxn FAM c/rxn FAM c/rxn FAM mmx1 mmx2 mmx3 mmx4 mmx5 mmx6 mmx7 mmx8 mmx9 PathID qpcr 10
11 DNA Master Mixes: Bovine Blood Extract avg Ct Eff %: 97% 106% 106% 110% 105% 111% 111% 142% 102% c/rxn FAM c/rxn FAM c/rxn FAM c/rxn FAM c/rxn FAM mmx1 mmx2 mmx3 mmx4 mmx5 mmx6 mmx7 mmx8 mmx9 PathID qpcr 11
12 DNA Master Mixes: Porcine Oral Fluid Extract avg Ct Eff %: 109% 99% 97% 104% 106% 111% 105% 122% 100% c/rxn FAM c/rxn FAM c/rxn FAM c/rxn FAM c/rxn FAM mmx1 mmx2 mmx3 mmx4 mmx5 mmx6 mmx7 mmx8 mmx9 PathID qpcr 12
13 DNA Master Mixes: Porcine Nasal Swab Extract avg Ct Eff %: 171% 110% 89% 112% 100% 106% 90% 140% 94% c/rxn FAM c/rxn FAM c/rxn FAM c/rxn FAM c/rxn FAM mmx1 mmx2 mmx3 mmx4 mmx5 mmx6 mmx7 mmx8 mmx9 PathID qpcr 13
14 Results by Consistency Counts: DNA qpcr (DNA) Master Mix DNA H 2 O Blood Ear Feces Oral Nasal mmx 1 + o + + o + mmx mmx mmx 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 Excellent + Good o Poor X Key Detects at least 7 of 8 replicates between copies/rxn. No Ct cutoff. Detects 100% of three replicates at 90 copies/rxn. Detects 0% of three replicates at the Good concentration. 14
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 10 sets: Solid = FAM channel = North American PRRSV target Hatched = VIC channel = European PRRSV target mmx2 = AgPath One-Step RT-PCR mmx3 = PathID Multiplex One-Step RT-PCR mmx1, mmx5 mmx11 = competitors master mixes 15
16 avg Ct %eff RNA Master Mixes: Water (control) 40.0 FAM = N. American PRRS; VIC = European PRRS NA 112% 93% 107% 88% 83% 106% 103% 95% 74% 113% EU 92% 101% 105% 108% 83% 104% 94% 101% 85% 56% c/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 mmx1 mmx2 mmx3 mmx5 mmx6 mmx7 mmx8 mmx9 mmx10 mmx11 AgPath PathID One-Step Multiplex RT-PCR One-Step RT-PCR 16
17 RNA Master Mixes: Bovine Feces Extract avg Ct %eff NA 84% 66% 94% 50% 29% 33% 92% 40% 67% 78% EU 94% 50% 93% 55% 21% 19% 55% 41% 71% #DIV/0! c/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 mmx1 mmx2 mmx3 mmx5 mmx6 mmx7 mmx8 mmx9 mmx10 mmx11 AgPath PathID One-Step Multiplex RT-PCR One-Step RT-PCR 17
18 RNA Master Mixes: Ear Notch Soak Extract avg Ct %eff 40.0 NA 102% 60% 94% 67% 57% 39% 65% 59% 70% 95% EU 76% 73% 88% 59% 56% 36% 57% 63% 78% 59% c/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 mmx1 mmx2 mmx3 mmx5 mmx6 mmx7 mmx8 mmx9 mmx10 mmx11 AgPath PathID One-Step Multiplex RT-PCR One-Step RT-PCR 18
19 RNA Master Mixes: Bovine Blood Extract avg Ct %eff NA 85% 78% 94% 78% 57% 42% 108% 63% 77% 64% EU 78% 76% 107% 76% 60% 75% 88% 84% 89% 49% c/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 mmx1 mmx2 mmx3 mmx5 mmx6 mmx7 mmx8 mmx9 mmx10 mmx11 AgPath PathID One-Step Multiplex RT-PCR One-Step RT-PCR 19
20 RNA Master Mixes: Porcine Oral Fluid Extract avg Ct %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% c/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 mmx1 mmx2 mmx3 mmx5 mmx6 mmx7 mmx8 mmx9 mmx10 mmx11 AgPath PathID One-Step Multiplex RT-PCR One-Step RT-PCR 20
21 avg Ct RNA Master Mixes: Porcine Nasal Swab Extract %eff NA 72% 53% 66% 49% 56% 36% 71% 36% 70% 50% EU 90% 49% 97% 43% 53% 63% 47% 48% 70% 46% c/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 mmx1 mmx2 mmx3 mmx5 mmx6 mmx7 mmx8 mmx9 mmx10 mmx11 AgPath PathID One-Step Multiplex RT-PCR One-Step RT-PCR 21
22 Results Summary: RNA RNA RT-PCR Master Mix H 2 O 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 o mmx 11 o o x x x Superior Excellent + Good o Poor x Key Detects at least 7 of 8 replicates between copies/rxn, for both assays of the multiplex. No Ct cutoff. Detects at least 6 of 7 between copies/rxn for both assays of the multiplex. Or one assay at Superior and one at Excellent. Detects 100% of three replicates at 47 copies/rxn for both assays of the multiplex. Or one assay at Excellent and one at Good. Detects 0% of three replicates at 47 copies/rxn for one or both assays of the multiplex. 22
23 Conclusions 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 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.
24 Thank you 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 24
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