Benchmarking of qPCR and qRT-PCR master mixes ... 2016/10/20  · Comparisons of qPCR...

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Transcript of Benchmarking of qPCR and qRT-PCR master mixes ... 2016/10/20  · Comparisons of qPCR...

  • The world leader in serving science

    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

    a v

    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

    a v

    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

    a v

    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

    a v

    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

    a v

    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

    a v

    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

  • 16

    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

    a v

    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.