DNA Molecular Detection of Mycoplasmas: Introducing Real-Time PCR

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DNA Molecular Detection of Mycoplasmas: Introducing Real-Time PCR Pablo Lopez, DVM, MBA IDEXX Laboratories,Inc., Westbrook, Maine USA 2010 IDEXX Laboratories, Inc. All rights reserved.

Polymerase Chain Reaction (PCR) 1986 First description of the polymerase chain reaction - Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Cold Spring Harb Smp Quant Biol 1986; 51 Pt 1:263-73 1988 First description of a thermostable DNA polymerase - Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Science 1988 Jan 29; 239(4839): 487-91 1993 First description of real-time polymerase chain reaction - Kinetic PCR analysis: real-time monitoring of DNA amplification reactions Higuchi R, Fockler C, Dollinger G, Watson R Biotechnology (NY) 1993 Sep; 11(9): 1026-30 1993 Nobel Prize in Chemistry - Kary B. Mullis Polymerase Chain Reaction - Michael Smith Site-directed mutagenesis 1996 First commercial real-time PCR instruments available 1 2010 IDEXX Laboratories, Inc. All rights reserved.

Pathogenic Avian Mycoplasmas: Background Pathogenic avian mycoplasmas - Mycoplasma gallisepticum: chickens and turkeys - Mycoplasma synoviae: chickens and turkeys - Mycoplasma meleagridis: turkeys - Mycoplasma iowae: turkeys Disease symptoms - Airsacculitis, sinusitis - Synovitis, arthritis - Skeletal deformities - Embryo mortality - Poor performance Diagnosis - Culture: gold standard - Antibody detection: Serum Plate Agglutination (SPA), Hemagglutination Inhibition (HI), ELISA - Molecular detection: PCR 2 2010 IDEXX Laboratories, Inc. All rights reserved.

M. gallisepticum, M. synoviae, M. meleagridis Real Time PCR Assay Overview Hybridization probes detection chemistry - Crossing point and T m data values Surveillance (Screening) Testing - Crossing point value for yes/no determination Confirmatory Testing - Crossing point value - T m value for additional data confirmation of correct target detection Sequences for primers and probes based on amplicon sequences from S. Kleven - Avian Diseases 53:103-107, 2009. Ziv Raviv and Stanley H. Kleven The Development of Diagnostic Real-Time TaqMan PCRs for the Four Pathogenic Avian Mycoplasmas 3 2010 IDEXX Laboratories, Inc. All rights reserved.

Hybridization Probes Detection Format Donor probe (D) provides energy to Acceptor probe (A) to emit signal when both probes are bound to target sequence (Fluorescence Resonance Energy Transfer) 3 primer probe 1 D A probe 2 D: Donor A: Acceptor 5 primer Probes are recycled throughout amplification program anneal Signals are collected by the instrument and used to calculate the crossing point values primer D 3 5 primer A probe 1 probe 2 Melting curves and T m values may be generated with hybridization probes 4 2010 IDEXX Laboratories, Inc. All rights reserved.

Cycling Program and Instrumentation M. gallisepticum, M. synoviae, M. meleagridis Real-Time PCR Instrumentation: LightCycler 480 with 96-well block Cycling Program: used for Mg, Ms and Mm real-time PCR assays - Activation: 1 cycle 95 C 10 minutes - Amplification: 45 cycles 95 C 20 seconds 60 C 20 seconds with a single fluorescence data acquisition 72 C 15 seconds - Melting Curve: 1 cycle 95 C 1 minute 45 C 1 minute 80 C continuous 0.14 C/second with collection of 4 fluorescence data acquisitions per second - Cooling: 1 cycle 40 C 30 seconds 5 2010 IDEXX Laboratories, Inc. All rights reserved.

Sample DNA Extraction Protocol used to generate data Tracheal Swabs and Mycoplasma Collection Broth High Pure PCR Template Preparation Kit (Roche Applied Science) -Spin column based format Trachael swabs: - Swabs were rinsed in 0.5 ml PBS - 400 ul PBS were used for the DNA extraction Mycoplasma Collection Broth: - 1 ml broth was centrifuged at 1000xg for 3 minutes - Pellet was resuspended in 200 ul PBS All DNA was stored at -80 C 5 µl Sample DNA was used per PCR assay reaction (total volume was 20 µl) 6 2010 IDEXX Laboratories, Inc. All rights reserved.

M. gallisepticum Amplicon Sequence Alignment with Wild Type mgc2 Sequences IDEXX Livestock and Poultry Diagnostics Consensus T T T T A T C C A G T A G T G G G T G C A G G T G C T G G G T T G A T T G T T G T T T C T T T A C T C T T G G G T T T A G G G A T T G G G A T T C C G A T C G C 10 20 30 40 50 60 70 80 Mg amplicon AY556304 AY556290 AY556284 AY556276 AY556264 AY556245 Consensus Mg amplicon AY556304 AY556290 AY556284 AY556276 AY556264 AY556245 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T T G G T T T A G G G A T T G G G A T T C C G A T C G C 29 T T T T A T C C A G T A G T G G G T G C A G G T G C T G G T T G A T T G T T G T T T C T T T A C T C T T G G T T T A G G G A T T G G G A T T C C G A T C G C 80 T T T T A T C C A G T A G T G G G T G C A G G T G C T G G T T G A T T G T T G T T T C T T T A C T C T T G G T T T A G G G A T T G G G A T T C C G A T C G C 80 T T T T A C C C A G T A G T G G G T G C A G G T G C T G G T T G A T C G T T G T T T C T T T A C T C T T G G T T T A G G G A T T G G G A T T C C G A T C G C 80 T T T T A C C C A G T A G T G G G C G C A G G T G C T G G T T G A T T G T T G T T T C T T T A C T C T T G G T T T A G G G A T T G G G A T T C C G A T C G C 80 T T T T A T C C A G T A G T G G G T G C A G G T G C T G G T T G A T T G T T G T T T C T T T A C T C T T G G T T T A G G G A T T G G G A T T C C G A T C G C 80 T T T T A T C C A G T A G T G G G T G C A G G T G C T G G T T G A T T G T T G T T T C T T T A C T C T T G G T T T A G G G A T T G G G A T T C C G A T C G C 80 T A A G A A A A A A G A A A G A A T G A T G A T C C A A G A A C G T G A A G A A C A C C A A A A G A T G G T T G A A T C C C T T G G A A T A A T C G A A G A A C 90 100 110 120 130 140 150 160 T A A G A A A A A A G A A A G A A T G A T G A T C C A A G A A C G T G A A G A A C A C C A A A A G A T G G T T G A A T C C C T T G G 95 T A A G A A A A A A G A A A G A A T G A T G A T C C A A G A A C G T G A A G A A C A C C A A A A G A T G G T T G A A T C C C T T G G A A T A A T C G A A G A A C 160 T A A G A A A A A A G A A A G A A T G A T G A T C C A A G A A C G T G A A G A A C A C C A A A A G A T G G T T G A A T C C C T T G G A A T A A T C G A A G A A C 160 T A A G A A A A A A G A A A G A A T G A T G A T C C A A G A A C G T G A A G A A C A C C A A A A G A T G G T T G A A T C C C T T G G A A T A A T C G A G C A A C 160 T A A G A A A A A A G A A A G A A T G A T G A T C C A A G A A C G T G A A G A A C A C C A A A A G A T G G T T G A A T C C C T T G G T A T A A T C G A A G A A C 160 T A A G A A A A A A G A A A G A A T G A T G A T C C A A G A A C G T G A A G A A C A C C A A A A G A T G G T T G A A T C C C T T G G A A T A A T T G A A G A A C 160 T A A G A A A A A A G A A A G A A T G A T G A T C C A A G A A C G T G A A G A A C A C C A A A A G A T G G T T G A A T C C C T T G G A A T A A T T G A A G A A C 160 AY556304 and AY556290: Chicken isolates AY556284 and Ay556276: Turkey isolates AY556264 and AY 556245: House Finch isolates All sequences from PDRC (N. Ferguson and S. Kleven) 7 2010 IDEXX Laboratories, Inc. All rights reserved.

M. synoviae Amplicon Sequence Alignment with Intergenic Spacer Region Sequences IDEXX Livestock and Poultry Diagnostics Consensus Ms amplicon AY768810 FM213418 FM213412 FM213408 AJ781002 Consensus Ms amplicon AY768810 FM213418 FM213412 FM213408 AJ781002 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C T T A C G G A G T A C A T T A W T T T T A C A A A A G G C A T T T T T T A T 100 110 120 130 140 150 160 170 180 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C C T C C T T T C T T A C G G A G T A C A T T A T T T T T A C A A A A G G C A T T T T T T A T 47 A A G T C G T A A C A A G G T A T C C C T A C G A G A A C G T G G G G A T G G A T T A C C T C C T T T C T T A C G G A G T A C A T T A T T T T T A C A A A A G G C A T T T T T T A T 180 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C T T A C G G A G T A C A T T A W T T T T A C A A A A G G C A T T T T T T A T 39 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C T T A C G G A G T A C A T T A W T T T T A C A A A A G G C A T T T T T T A T 39 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C T T A C G G A G T A C A T T A W T T T T A C A A A A G G C A T T T T T T A T 39 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C T T A C G G A G T A C A T T A W T T T T A C A A A A G G C A T T T T T T A T 39 T A A C T G A A A G C T T T T A G A T T T - - - T T C T A A A A G C G G T T G T G T A T C G C T T T T T T T X G C C T T G G G C T A T T G T A T T T A G T T T T G A G A G A A C A A 190 200 210 220 230 240 250 260 270 T A A C T G A A A G C T T T T A G A T T T - - - T T C T A A A A G C G G T T G T G T A T C G C T T T T T T T - G C C T T G G G C T A T T G T A T T T A G 119 T A A C T G A A A G C T T T T A G A T T T - - - T T C T A A A A G C G G T T G T G T A T C G C T T T T T T T - G C C T T G G G C T A T T G T A T T T A G T T T T G A G A G A A C A A 266 T A A C T G A A A G C T T T T A G A T T T - - - T T C T A A A A G C G G T T G T G T A T C G C T T T T T T T T G C C T T G G G C T A T T G T A T T T A G T T T T G A G A G A A C A A 126 T A A C T G A A A G C T T T T A G A T T T - - - T T C T A A A A G C G G T T G T G T A T C G C T T T T T T T T G C C T T G G G C T A T T G T A T T T A G T T T T G A G A G A A C A A 126 T A A C T G A A A G C T T T T A G A T T T - - - T T C T A A A A G C G G T T G T G T A T C G C T T T T T T T T G C C T T G G G C T A T T G T A T T T A G T T T T G A G A G A A C A A 126 T A A C T G A A A G C T T T T A G A K W A A A T T C T A A A A G C G G T T G T G T A T C G C T T T T T T T - G C C T T G G G C T A T T G T A T T T A G T T T T G A G A G A A C A A 128 AY768810: USDA FM213418: University of Liverpool (J. Bradbury) FM213412: University of Liverpool (J. Bradbury) FM213408: University of Liverpool (J. Bradbury) AJ781002: University of Liverpool (J. Bradbury) 8 2010 IDEXX Laboratories, Inc. All rights reserved.

M. meleagridis Amplicon Sequence Alignment with Intergenic Spacer Region Sequences IDEXX Livestock and Poultry Diagnostics Consensus - - - - - - - - - A A C A A G G T A T C C C T A C G A G A A C G T G G G G A T G G A T T A C C T C C T T T C T A C G G A G T A C A T T A G T T A A T T T A T T T 90 100 110 120 130 140 150 160 Mm amplicon AY762641 AJ780999 Consensus Mm amplicon AY762641 AJ780999 - - - - - - - - - A A C A A G G T A T C C C T A C G A G A A C G T G G G G A T G G A T T A C C T C C T T T C T A C G G A G T A C A T T A G T T A A T T T A T T T 71 T T A A G T C G T A A C A A G G T A T C C C T A C G A G A A C G T G G G G A T G G A T T A C C T C C T T T C T A C G G A G T A C A T T A G T T A A T T T A T T T 160 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C T A C G G A G T A C A T T A G T T A A T T T A T T T 27 A A T C A T T G G G A C T A A A A T T A A C C T T T A A G C C T G T T A T T T T A A T A T T G T C A T G A C T T G G T T T A A G G C T C T G A G T X A T A T A T 170 180 190 200 210 220 230 240 A A T C A T T G G G A C T A A A A T T A A C C T T T A A G C C T G T T A T T T T A A T A T T G T C A T G A C T T G G T T T A A G G C T C T G A G 143 A A T C A T T G G G A C T A A A A T T A A C C T T T A A G C C T G T T A T T T T A A T A T T G T C A T G A C T T G G T T T A A G G C T C T G A G T G A T A T A T 240 A A T C A T T G G G A C T A A A A T T A A C C T T T A A G C C T G T T A T T T T A A T A T T G T C A T G A C T T G G T T T A A G G C T C T G A G T T A T A T A T 107 AY762641: USDA AJ780999: University of Liverpool (J. Bradbury) 9 2010 IDEXX Laboratories, Inc. All rights reserved.

Analytical Sensitivity and Specificity 10 2010 IDEXX Laboratories, Inc. All rights reserved.

M. gallisepticum Real-Time PCR Analytical Sensitivity M. gallisepticum 100 pg 10 pg 1 pg 100 fg 10 fg 1 fg 0.1 fg NC Dilution CP Value T m value C 10 7 copies 15.29 62.55 10 6 copies 18.73 62.55 10 5 copies 22.44 62.55 10 4 copies 25.60 62.55 10 3 copies 28.90 62.55 10 2 copies 32.53 62.55 10 1 copies 35.91 62.55 nc negative negative 11 2010 IDEXX Laboratories, Inc. All rights reserved.

M. synoviae Real-Time PCR Analytical Sensitivity Sample CP Value T m value 10 7 copies 17.08 68.91 10 6 copies 20.55 68.81 10 5 copies 24.02 68.73 10 4 copies 27.51 68.67 10 3 copies 30.87 68.71 10 2 copies 34.23 68.67 10 1 copies 37.41 68.66 nc negative negative 12 2010 IDEXX Laboratories, Inc. All rights reserved.

M. meleagridis Real-Time PCR Analytical Sensitivity Sample CP Value T m value 10 6 copies 19.29 70.90 10 5 copies 22.86 70.90 10 4 copies 26.16 70.89 10 3 copies 29.69 70.88 10 2 copies 33.22 70.77 10 1 copies 36.77 70.90 nc negative negative 13 2010 IDEXX Laboratories, Inc. All rights reserved.

Analytical Specificity M. gallisepticum, M. synoviae, M. meleagridis M. gallisepticum M. synoviae M. meleagridis Sample DNA Crossing T m Crossing T m Crossing T m Point Point Point M. iowae negative negative negative negative negative negative M. lipofaciens negative negative negative negative negative negative M. cloacale negative negative negative negative negative negative M. imitans negative negative negative negative negative negative M. gallisnarum negative negative negative negative negative negative M. gallinaceum negative negative negative negative negative negative M. glycophilum negative negative negative negative negative negative M. pullorum negative negative negative negative negative negative M. gallopavonis negative negative negative negative negative negative 10 fg 28.58 63.99 28.92 68.48 28.71 71.48 1 fg 31.70 63.64 32.22 67.92 32.84 71.42 nc negative negative negative negative negative negative nc negative negative negative negative negative negative Non-pathogenic Mycoplasma DNA received from PDRC, University of Georgia All DNA tested negative with the three assays Assays did not recognize the non-pathogenic avian Mycoplasma DNA 14 2010 IDEXX Laboratories, Inc. All rights reserved.

Reproducibility 15 2010 IDEXX Laboratories, Inc. All rights reserved.

M. gallisepticum Real-Time PCR Inter-assay and Intra-assay Performance IDEXX Livestock and Poultry Diagnostics Assay 1 Assay 2 Assay 3 Dilution Crossing Crossing Crossing Crossing Crossing Crossing Crossing Crossing Crossing Standard Point Point Point Point Point Point Point Point Point Deviation Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 10 pg 18.59 18.62 18.60 18.64 18.69 18.65 18.57 18.55 18.49 0.059 1 pg 21.94 22.08 21.93 22.09 22.09 21.96 21.87 21.81 21.75 0.124 100 fg 25.17 25.47 25.30 25.33 25.25 25.23 25.20 25.13 25.03 0.126 10 fg 28.68 28.75 28.73 28.74 28.70 28.68 28.63 28.64 28.05 0.219 1 fg 31.95 31.84 31.95 31.72 31.87 31.88 31.85 32.06 31.85 0.094 nc negative negative negative negative negative negative negative negative negative Assay 1 Assay 2 Assay 3 Dilution T m T m T m T m T m T m T m T m T m Standard Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Deviation 10 pg 64.52 64.42 64.14 64.11 64.01 64.02 63.97 63.99 63.94 0.207 1 pg 64.07 64.07 64.00 63.97 63.92 63.89 63.90 63.94 63.88 0.073 100 fg 64.10 63.95 63.84 63.80 63.75 63.70 63.90 63.91 63.85 0.118 10 fg 63.75 63.71 63.70 63.12 63.12 63.06 63.75 63.81 63.71 0.321 1 fg 63.11 63.09 62.96 63.18 63.06 63.08 63.49 63.56 63.57 0.238 nc negative negative negative negative negative negative negative negative negative negative Standard Deviation is < 3% indicating acceptable inter-assay performance Target was M. gallisepticum plasmid ten-fold dilution series 16 2010 IDEXX Laboratories, Inc. All rights reserved.

M. synoviae Real-Time PCR Inter-assay and Intra-assay Performance Assay 1 Assay 2 Assay 3 Dilution Crossing Crossing Crossing Crossing Crossing Crossing Crossing Crossing Crossing Standard Point Point Point Point Point Point Point Point Point Deviation Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 10 pg 18.72 18.70 18.68 18.65 18.63 18.63 18.71 18.63 18.58 0.046 1 pg 22.19 22.24 22.17 22.10 22.11 22.06 22.08 22.05 22.00 0.076 100 fg 25.73 25.66 25.78 25.78 25.71 25.70 25.75 25.70 25.70 0.040 10 fg 29.14 29.20 29.12 29.13 29.05 29.09 29.13 29.00 29.02 0.064 1 fg 32.68 32.44 32.62 32.58 32.61 32.60 32.59 32.61 32.45 0.079 nc negative negative negative negative negative negative negative negative negative Assay 1 Assay 2 Assay 3 Dilution T m T m T m T m T m T m T m T m T m Standard Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Deviation 10 pg 69.10 69.06 69.05 69.34 69.21 69.14 69.32 69.21 69.14 0.105 1 pg 69.08 68.91 68.89 69.23 69.04 68.96 69.25 69.07 68.99 0.128 100 fg 69.08 68.91 68.90 69.17 68.97 68.94 69.24 69.05 69.00 0.118 10 fg 69.16 69.07 69.07 69.18 69.06 69.05 69.34 69.19 69.19 0.094 1 fg 68.27 69.03 68.15 69.06 68.04 68.04 68.33 68.19 68.19 0.396 nc negative negative negative negative negative negative negative negative negative Standard Deviation is < 3% indicating acceptable inter-assay performance Target was M. synoviae plasmid ten-fold dilution series 17 2010 IDEXX Laboratories, Inc. All rights reserved.

M. meleagridis Real-Time PCR Inter-assay and Intra-assay Performance Assay 1 Assay 2 Assay 3 Dilution Crossing Crossing Crossing Crossing Crossing Crossing Crossing Crossing Crossing Standard Point Point Point Point Point Point Point Point Point Deviation Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 10 pg 19.43 19.42 19.24 19.47 19.49 19.48 19.45 19.47 19.45 0.076 1 pg 22.92 22.81 22.79 23.00 22.96 22.88 22.81 22.99 22.91 0.080 100 fg 26.22 26.42 26.15 26.49 26.32 26.34 26.18 26.29 26.18 0.117 10 fg 29.76 29.71 29.70 29.89 29.76 29.75 29.66 29.69 29.69 0.068 1 fg 33.16 33.20 33.09 33.31 33.52 33.28 32.85 32.95 32.90 0.217 nc negative negative negative negative negative negative negative negative negative Assay 1 Assay 2 Assay 3 Standard Dilution T m T m T m T m T m T m T m T m T m Deviation Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 10 pg 70.23 70.09 70.00 70.27 70.15 70.04 71.14 71.21 71.31 0.553 1 pg 70.20 70.07 70.03 70.23 70.10 70.05 71.00 71.08 71.21 0.499 100 fg 70.27 70.17 70.14 70.30 70.21 70.16 70.96 70.01 71.18 0.405 10 fg 70.26 70.17 70.11 70.27 70.19 70.15 71.07 71.09 71.18 0.464 1 fg 70.25 70.09 70.01 70.28 70.12 70.05 71.04 71.01 71.12 0.470 nc negative negative negative negative negative negative negative negative negative Standard Deviation is < 3% indicating acceptable inter-assay performance Target was M. meleagridis plasmid ten-fold dilution series 18 2010 IDEXX Laboratories, Inc. All rights reserved.

Performance 19 2010 IDEXX Laboratories, Inc. All rights reserved.

M. gallisepticum Real-Time PCR Sample Crossing T m Point Mg-1 31.24 64.26 Mg-2 25.46 64.28 Mg-3 negative negative Mg-4 24.73 63.90 Mg-5 negative negative Mg-6 31.83 62.71 DNA samples from chickens M. gallisepticum DNA positive 10 3 copies 28.64 63.83 10 2 copies 31.74 63.39 NC negative negative 20 2010 IDEXX Laboratories, Inc. All rights reserved.

M. gallisepticum Real-Time PCR Sample Crossing T m Sample Crossing T m Point Point Sample 1 negative negative Sample 17 negative negative Sample 2 negative negative Sample 18 negative negative Sample 3 negative negative Sample 19 negative negative Sample 4 negative negative Sample 20 negative negative Sample 5 negative negative Sample 21 negative negative Sample 6 negative negative Sample 22 negative negative Sample 7 negative negative Sample 23 negative negative Sample 8 negative negative Sample 24 negative negative Sample 9 negative negative Sample 25 negative negative Sample 10 negative negative Sample 26 negative negative Sample 11 negative negative Sample 27 negative negative Sample 12 negative negative Sample 28 negative negative Sample 13 negative negative Sample 29 negative negative Sample 14 negative negative Sample 30 negative negative Sample 15 negative negative Sample 31 negative negative Sample 16 negative negative Sample 32 negative negative DNA Samples from chickens M. gallisepticum negative 10 fg 28.60 63.83 nc negative negative 1 fg 31.82 63.39 nc negative negative 21 2010 IDEXX Laboratories, Inc. All rights reserved.

M. gallisepticum Real-Time PCR DNA samples from turkeys M. gallisepticum DNA positive DNA samples from turkeys M. gallisepticum DNA negative Sample Crossing T m Sample Crossing T m Point Point TK-1 24.20 63.02 Tk-11 negative negative TK-2 29.01 62.79 Tk-12 negative negative TK-3 31.53 62.64 Tk-13 negative negative TK-4 31.02 62.73 Tk-14 negative negative TK-5 31.71 62.67 Tk-15 negative negative TK-6 25.23 62.79 Tk-16 negative negative TK-7 26.26 62.81 Tk-17 negative negative TK-8 26.77 62.85 Tk-18 negative negative TK-9 30.99 62.73 Tk-19 negative negative TK-10 23.66 62.92 Tk-20 negative negative Tk-21 negative negative 10 fg 28.65 63.60 Tk-22 negative negative 1 fg 31.98 63.39 Tk-23 negative negative nc negative negative Tk-24 negative negative nc negative negative Tk-25 negative negative 10 fg 28.70 63.69 1 fg 31.84 63.16 nc negative negative nc negative negative 22 2010 IDEXX Laboratories, Inc. All rights reserved.

M. synoviae Real-Time PCR DNA samples from chickens M. synoviae DNA positive Sample Crossing T m Sample Crossing T m Sample Crossing T m Point Point Point MS CH 1 32.30 68.46 MS CH 26 28.54 67.46 MS CH 51 26.29 67.23 MS CH 2 29.01 67.61 MS CH 27 34.54 68.31 MS CH 52 31.91 68.04 MS CH 3 33.61 68.32 MS CH 28 29.93 67.58 MS CH 53 27.86 67.38 MS CH 4 27.45 67.62 MS CH 29 30.05 67.55 MS CH 54 24.57 67.29 MS CH 5 32.05 67.59 MS CH 30 28.58 67.47 MS CH 55 29.69 67.25 MS CH 6 31.86 67.57 MS CH 31 31.48 68.15 MS CH 56 26.50 67.39 MS CH 7 negative negative MS CH 32 29.09 67.45 MS CH 57 26.75 67.46 MS CH 8 negative negative MS CH 33 25.16 67.54 MS CH 58 26.86 67.40 MS CH 9 32.92 67.63 MS CH 34 33.82 68.06 MS CH 59 24.54 67.30 MS CH 10 32.71 68.15 MS CH 35 24.86 67.46 MS CH 60 24.69 67.52 MS CH 11 32.74 67.43 MS CH 36 26.56 67.55 MS CH 61 29.63 67.47 MS CH 12 27.65 67.64 MS CH 37 31.62 67.46 MS CH 62 28.05 67.36 MS CH 13 27.02 67.50 MS CH 38 23.49 67.54 MS CH 63 24.13 67.33 MS CH 14 28.34 67.42 MS CH 39 25.69 67.42 MS CH 64 26.34 67.36 MS CH 15 26.66 57.41 MS CH 40 24.93 67.48 MS CH 65 30.62 67.50 MS CH 16 28.00 67.53 MS CH 41 27.05 67.45 MS CH 66 24.95 67.35 MS CH 17 31.01 67.56 MS CH 42 25.69 67.23 MS CH 67 25.46 67.31 MS CH 18 28.19 67.44 MS CH 43 24.72 67.28 MS CH 68 25.96 67.41 MS CH 19 27.47 67.42 MS CH 44 27.32 67.38 MS CH 69 30.20 67.41 MS CH 20 35.06 68.19 MS CH 45 24.55 67.36 MS CH 70 27.49 67.30 MS CH 21 31.72 67.53 MS CH 46 25.14 67.29 MS CH 22 35.60 67.83 MS CH 47 26.01 67.29 10 fg 30.92 68.34 MS CH 23 37.76 68.76 MS CH 48 28.52 67.39 1 fg 34.79 68.32 MS CH 24 34.52 68.09 MS CH 49 26.11 67.46 nc negative negative MS CH 25 29.04 67.63 MS CH 50 23.61 67.30 nc negative negative 23 2010 IDEXX Laboratories, Inc. All rights reserved.

M. synoviae Real-Time PCR Sample Crossing T m Point MS 1 negative negative MS 2 negative negative MS 3 negative negative MS 4 negative negative MS 5 negative negative MS 6 negative negative MS 7 negative negative MS 8 negative negative MS 9 negative negative MS 10 negative negative MS 11 negative negative MS 12 negative negative MS 13 negative negative MS 14 negative negative MS 15 negative negative MS 16 negative negative DNA Samples from chickens M. synoviae negative nc negative negative 10 fg 30.86 67.91 1 fg 35.00 68.05 24 2010 IDEXX Laboratories, Inc. All rights reserved.

M. synoviae Real-Time PCR Sample Crossing T m Sample Crossing T m Point Point MS TK 1 negative negative MS TK 32 negative negative MS TK 2 negative negative MS TK 33 negative negative MS TK 3 negative negative MS TK 34 negative negative MS TK 4 negative negative MS TK 35 negative negative MS TK 5 negative negative MS TK 36 negative negative MS TK 6 negative negative MS TK 37 negative negative MS TK 7 negative negative MS TK 38 negative negative MS TK 8 negative negative MS TK 39 negative negative MS TK 9 negative negative MS TK 40 negative negative MS TK 10 negative negative MS TK 41 negative negative MS TK 11 negative negative MS TK 42 negative negative MS TK 12 negative negative MS TK 43 negative negative MS TK 13 negative negative MS TK 44 negative negative MS TK 14 negative negative MS TK 45 negative negative MS TK 15 negative negative MS TK 46 negative negative MS TK 16 negative negative MS TK 47 negative negative MS TK 17 negative negative MS TK 48 negative negative MS TK 18 negative negative MS TK 49 negative negative MS TK 19 negative negative MS TK 50 negative negative MS TK 20 negative negative MS TK 51 negative negative MS TK 21 negative negative MS TK 52 negative negative MS TK 22 negative negative MS TK 53 negative negative MS TK 23 negative negative MS TK 54 negative negative MS TK 24 negative negative MS TK 55 negative negative MS TK 25 negative negative MS TK 56 negative negative MS TK 26 negative negative MS TK 57 negative negative MS TK 27 negative negative MS TK 28 negative negative 10 fg 30.00 68.32 MS TK 29 negative negative 1 fg 30.00 negative MS TK 30 negative negative nc negative negative MS TK 31 negative negative nc negative negative DNA Samples from turkeys M. synoviae negative 25 2010 IDEXX Laboratories, Inc. All rights reserved.

M. meleagridis Real-Time PCR Sample Crossing T m Point Sample 1 21.80 69.74 Sample 13 19.75 69.74 Sample 25 23.20 69.27 Sample 37 21.03 69.74 Sample 49 20.45 69.74 Sample 61 22.93 69.47 Sample 73 24.88 69.43 Sample 85 18.75 69.72 DNA Samples from turkeys M. Meleagridis positive 1 pg 25.93 69.93 100 fg 29.85 69.94 nc negative negative 26 2010 IDEXX Laboratories, Inc. All rights reserved.

M. meleagridis Real-Time PCR Sample Crossing T m Sample Crossing T m Point Point MM TK 1 negative negative MM TK 32 negative negative MM TK 2 negative negative MM TK 33 negative negative MM TK 3 negative negative MM TK 34 negative negative MM TK 4 negative negative MM TK 35 negative negative MM TK 5 negative negative MM TK 36 negative negative MM TK 6 negative negative MM TK 37 negative negative MM TK 7 negative negative MM TK 38 negative negative MM TK 8 negative negative MM TK 39 negative negative MM TK 9 negative negative MM TK 40 negative negative MM TK 10 negative negative MM TK 41 negative negative MM TK 11 negative negative MM TK 42 negative negative MM TK 12 negative negative MM TK 43 negative negative MM TK 13 negative negative MM TK 44 negative negative MM TK 14 negative negative MM TK 45 negative negative MM TK 15 negative negative MM TK 46 negative negative MM TK 16 negative negative MM TK 47 negative negative MM TK 17 negative negative MM TK 48 negative negative MM TK 18 negative negative MM TK 49 negative negative MM TK 19 negative negative MM TK 50 negative negative MM TK 20 negative negative MM TK 51 negative negative MM TK 21 negative negative MM TK 52 negative negative MM TK 22 negative negative MM TK 53 negative negative MM TK 23 negative negative MM TK 54 negative negative MM TK 24 negative negative MM TK 55 negative negative MM TK 25 negative negative MM TK 56 negative negative MM TK 26 negative negative MM TK 57 negative negative MM TK 27 negative negative MM TK 28 negative negative 10 fg 29.76 70.26 MM TK 29 negative negative 1 fg 33.20 70.09 MM TK 30 negative negative nc negative negative MM TK 31 negative negative nc negative negative DNA Samples from turkeys M. meleagridis negative 27 2010 IDEXX Laboratories, Inc. All rights reserved.

M. gallisepticum, M. synoviae, M. meleagridis Real-Time PCR Summary Sensitivity: Analytical sensitivity limit of detection is 10 copies of target sequence Specificity: Non-pathogenic Mycoplasma are not detected Data: - Crossing Point (CP): value indicates presence of target DNA - Tm: value confirms correct target sequence detected Clinical performance: Assays performed as expected - Mycoplasma negative chicken and turkey flocks were correctly identified - Mycoplasma positive chicken and turkey flock were correctly identified 28 2010 IDEXX Laboratories, Inc. All rights reserved.

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