1

The world leader in serving science

MycoSEQTM Kits for Lot Release Testing Applications

As a Limit Test for Impurities

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Mycoplasma Testing for Cell Culture Produced and Cell Based Therapeutics

The Challenge

• Mammalian cell cultures used for manufacture of therapeutics must be tested

for Mycoplasma and be free of Mycoplasma

• In the past, the test for Mycoplasma accepted by regulatory agencies was

a 28-day culture based test

• Long testing cycles lead to delays in lot disposition

• Cell or Tissue Therapy products have short shelf life, require rapid test methods.

• The 28-day test requires specialized expertise, live control organisms and is generally

done by specialty contract testing labs at a very high cost per sample

The Alternative • Replace the traditional 28-day Mycoplasma test with a rapid qPCR based test.

Results in hours or days

The Solution • MycoSEQ™ kits: rapid, sensitive qPCR Mycoplasma Detection

Accepted by the FDA and EMA as an alternative to the 28-day Lot Release test for

Mycoplasma

MycoSEQTM kits were specifically designed to meet the expectations for

nucleic acid based tests for mycoplasma lot release testing

3

Critical Considerations:

qPCR for Mycoplasma Testing

Key Attribute MycoSEQ Feature

Intended Use • Specifically Designed for Lot Release testing

Sensitivity

• Consistent across species: 1-3 Genome Copies/PCR

reaction

• Sensitivity not dependant on metabolic state of

Mycoplasma, 1 cell contains 1 genome copy

Specificity • Specific for Mycoplasma

• Proven specificity in multiple studies

Results Interpretation

• Objective by using acceptance criteria

• Cycle at Threshold (Ct): Input DNA quantity

• Melting Temperature (Tm): Amplicon Size

• Derivative Value (DV): Amplicon Quantity

Controls

• Discriminate positive control cross-contamination to real

Mycoplasma positive

• PCR Inhibition Control

• Negative Control

• Positive Control

Support

• Drug Master File in place with FDA

• Specialized Field Application Scientists: On-site training

• Experience with validation design and regulatory support

• Equipment Validation services: IQ/OQ

4

Regulatory Guidance

European Pharmacopoeia

Specificity

Inclusivity and exclusivity (e.g.,

Clostridium, Lactobacillus,

Streptococcus)

Limit of Detection

• 10 CFU or copy equivalent/ml as

alternative to culture method

• 100 CFU or copy equivalent/ml as

alternative to indicator cell culture

method

Robustness

Deliberate variations, e.g., reagent

volumes or collaborative studies

“Nucleic Acid Amplification Techniques

(NAT) may be used as an alternative

after suitable validation.”

Outline for Validation

Species Selection Tests

5

2 Options for Rapid Mycoplasma Detection

The Challenge

• Concentration and extraction of

Mycoplasma directly from test sample

• 3-5 hour time to result

• Sensitivity validated at less than 10

CFU/mL from 10 mL sample

• Sample preparation protocols optimized to

enable high sensitivity detection from high

titer/low viability bioreactor samples

Direct qPCR

• Inoculation of cell culture sample into

Mycoplasma broth

• Incubate for 3-7 days, test by MycoSEQ™

• Sensitivity validated at less than 10

CFU/mL from 10 mL sample

• Ideal when a wide variety of bioreactor

samples require a single test protocol

Enrichment Culture Followed

by qPCR

Both approaches have been validated, filed and accepted

by regulatory authorities

6

Risk Assessment for Rapid Mycoplasma Testing

In-Process

Testing

• In-process testing by qPCR monitors for the presence of Mycoplasma

throughout cell culture processes.

• Offsets risk of a single qPCR test at bioreactor harvest as the only data

point.

Raw

Materials

• Are there any raw materials used in the process present a risk for

Mycoplasma?

• Can this risk be mitigated?

• At what point are they introduced into the process?

Viral Filtration/

Viral Inactivation

• What measures are used?

• Are these steps effective in inactivating or removing Mycoplasma?

• Is there any risk in the process of undetected Mycoplasma carrying

through purification?

Cell Associated

Mycoplasma

• As cell associated Mycoplasma are more of a risk in cell banking, review

procedures for testing during cell banking. Mycoplasma free?

• Is there a risk of Mycoplasma introduced into the bioreactor associating

with mammalian cells at significant levels? Reactor stirring etc.

• Sample preparation for qPCR: cells and media or media only?

7

Mycoplasma:

The relationship between a Colony Forming Unit (CFU) and a Genome Copy (GC)

Mycoplasma Cell

Mycoplasma chromosome, 1 circular

chromosome per cell, 650Kbp -1900Kbp

16S rRNA gene, the target of the

MycoSEQ™ assay

Additional copy of 16S rRNA gene,

Some species have 2 copies per genome

Ideally, 1 GC equals 1 CFU, but that must

be verified. If the ratio changes because the

sample or stock has cells that contain DNA,

but do not grow and represent as a CFU….

The sensitivity of an NAT test may be misleading…

8

Genome Copy (GC)/Colony Forming Unit (CFU) Assessment A Critical Tool in Validation

• Mycoplasma arginini culture sample

• Titer of sample: 4 CFU/mL

• Ct of sample by qPCR = 30.6

• Ct of 30.6 ~10 GC

• PCR test Vol. = 1.3 mL test sample

• 4 CFU~8 GC

Standard Curve Generated by qPCR Analysis

of Purified Mycoplasma arginini DNA

qPCR is the only technology that enables this critical measurement

The regulatory expectation is that

live Mycoplasma stocks used in

validation have low GC/CFU ratios

For Mycoplasma arginini, in this

experiment, the ratio is~ 2 GC/CFU

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Applications of Ct Using the qPCR Assays

• Viability assessment

• Acceptance criteria for test and control samples

• Estimation of contamination level in positive samples

• Comparison of results between experiments

• GC/CFU ratio

• Sample preparation protocol optimization

• Validation Studies - Compare your Ct to expected values for each species

- Estimation of LLOD by extrapolation from values obtained at 10 GC or CFU/sample.

The Cycle at Threshold (Ct) is a quantitative parameter representing the quantity

of DNA present in the test sample at the start of the PCR process

10

qPCR Has Enabled New Understanding of Mycoplasma Biology

• Mycoplasma growth rates measured by qPCR show rapid growth in liquid media

• Stationary phase is reached quickly, followed by a decline in viability

• The GC/CFU ratio is aligned in log phase growth, but diverges as viability declines.

• qPCR sensitivity is not impacted by viability loss

• If CFU assessment (agar plating) is not done at the appropriate time point, culture-based test

results could be very misleading.

A. Dabrazhynetskaya et al. Journal of Applied

Microbiology 111, 904–914

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3 Components of MycoSEQ™ Assay Sensitivity

Background

Reduction and

Mycoplasma

Concentration

Large Volume

Protocols for

High Percentage

Nucleic Acid

Recovery

Detection Using

Highest Sensitivity

qPCR Assay

Highest

Sensitivity

Mycoplasma

Detection

• Bioreactor

Harvest

• Cell Therapy

• Tissue Therapy

• Raw Materials

• Media

• Serum

PrepSEQTM Magnetic

Bead Nucleic Acid

Preparation

• Highest

percentage

recovery

• Manual or

Automated

options

MycoSEQ™

Detection Assay

• 31 Mycoplasma

specific primers

• 1-3 copy per

PCR reaction

sensitivity

• Consistent

across species

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Assay Sensitivity as a Function of Test Sample Volume

Starting

Sample

Volume

Sample

Prep

Elution Vol.

Assay

Sensitivity*

(GC/Rxn)

Sample Volume

(PCR Reaction)

Sensitivity

(CFU or

GC/mL)

10 mL 100 uL 10 1 mL 10

1 mL 100 uL 10 100 uL 100

100 uL 100uL 10 10 uL 1000

*The assay sensitivity stated above is not the lower limit of detection of the assay.

We recommend that validation experiments are done at 10 GC or CFU/mL

in order to obtain consistent results

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Mycoplasma Assay Sensitivity

Mycoplasma arginini

105 104 103 102 10 1

Analysis of a 10-fold dilution Series

Of Purified M.Arginini DNA:

Melt Analysis at 1 GC/Rxn

• LOD: <10 copy/reaction

• Tm: ~80°C

• Same LOD with ATCC DNA

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Validation Approach

Limit of Detection

ICH Q2(R1) Requires

Validation of Limit of

Detection for Limit Tests

for Impurities

Sensitivity of

MycoSEQ assay

We Recommend

Qualification and Validation

at an LOD of 10 GC/mL

• This does not mean

Lowest Limit of

Detection (LLOD)

• EP Guidance

recommends validated

sensitivity of 10 GC/mL

• LLOD: 1 to 3 Genome

Copies/PCR reaction,

species dependent, A.

laidlawii is least sensitive

at 3 GC/mL from 10 mL

sample

• We do not recommend

validation testing at the

LLOD due to technical

challenges in validating

any limit test at this level

• Because MycoSEQ™ kits

utilize qPCR technology,

the LLOD can be

assessed using Ct values

obtained based on

principles of qPCR

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Discriminatory Positive / Extraction Control

• Positive control DNA made with

Mycoplasma amplicon modified to

have melting temperature (Tm) outside

range of Mycoplasma amplicons

• Allows additional level of confirmation

of positive test results

• Higher Tm allows discrimination

between true Mycoplasma and

accidental contamination of test

sample with Positive Control.

• Enables simple extraction control

spiking of test samples

• Can be used as a surrogate for

Mycoplasma DNA during method

qualification

Mycoplasma Discriminatory

Control

M. arginini

Original

Control

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Validation Recommendations

• Test 10 mL samples

• Optimize sample preparation protocol using test samples to be included in scope

of validation using Discriminatory Positive Control (DPC) DNA or purified Mycoplasma DNA

(M. arginini and A. laidlawii) at 10 GC/mL (100 GC/10 mL sample)

• Generate qualification data using purified Mycoplasma DNA or live Mycoplasma at 10 GC

or CFU/mL. Both options can also be used. Recommend all E.P. species

• Test a limited number (1-3 samples/species) spiked at 10 CFU/mL in PTC test to assess

comparability. 3 potential outcomes:

- Positive by PCR, Positive by PTC

- Positive by PCR, Negative by PTC

- Negative by PCR, Positive by PTC

• Analyze data, compile qualification report, design validation study, draft validation protocol

• Request meeting with relevant regulatory authorities to discuss qualification results,

validation expectations and validation study design. It is critical to address all

regulatory concerns in validation. For FDA, either Type C or Prior Approval

Supplement approach can be used here

• Execute validation study

Lot Release Testing as a Potential Replacement for the PTC test

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Validation Study

Potential Design

Specificity LOD, Part 1 LOD, Part 2

Part 1:

No detection of non-

Mycoplasma bacteria.

Data in DMF may be

acceptable

Part 2:

No interference from

customer sample matrix

(also addressed in LOD,

Part 1)

• 6 samples with 100 copies

(10 GC/mL) purified

Mycoplasma DNA spiked into

the lysate from 10 mL

samples

• Run 4 PCR reactions per

extractions (24 total PCR

reactions)

• 6 samples with 100 CFU (10

CFU/mL) live Mycoplasma

spiked directly into 10 mL

samples

• Run 4 PCR reactions

per extractions

(24 total PCR reactions)

For each Mycoplasma

species included

in the study,

This section may be optional

depending on discussion

with regulators. For each

Mycoplasma species

included in the study:

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Data and Results Comparison Example

10 mL test samples, spiked with M. arginini, DNA or Organisms

Mycoplasma

Species

(Type Strain)

Total Number

Tests/Positive Reactions % Positive

Mean CT

(n=24)

M. arginini G230T 24/24 100 30.90

Results from Mycosafe Validation, 10 GC/mL:

Mycoplasma

Species

(Type Strain)

Total Number

Tests/Positive Reactions % Positive

Mean CT

(n=4)

M. arginini G230T 4/4 100 32.8

Results from Mycosafe Validation, 10 CFU/mL:

Results from Customer Validation, 8 GC/mL:

Mycoplasma

Species

(Type Strain)

Total Number

Tests/Positive Reactions % Positive

Mean CT

(n=24)

M. arginini 24/24 100 31.1

Mycoplasma

Species

(Type Strain)

Total Number

Tests/Positive Reactions % Positive

Mean CT

(n=24)

M. arginini 24/24 100 31.5

Results from Customer Validation, 8 CFU/mL:

Mycoplasma

stocks and

DNA prepared

by Mycosafe

Mycoplasma

stocks and DNA

prepared by

Bionique

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Results Comparison: M. Arginini

• Analysis of 10-fold dilution series, purified M. arginini genomic DNA

• 10 GC/PCR reaction, Ct = 30.5

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Results from External Validation Study

Level of Detection, Part 1, 10 GC/mL from 10 mL sample: Summary of Results

Mycoplasma

Species

(Type Strain)

Total Number

Tests/Positive

Reactions

% Positive Mean CT

(n=24) SD CV (%)

A. laidlawii PG8T 24/24 100 33.87 0.625 1.8

M. arginini G230T 24/24 100 30.90 0.99 3.2

M. fermentans PG18T 24/24 100 32.21 1.68 5.2

M. hominis PG21T 24/24 100 29.53 0.86 2.9

M. hyorhinis BTS7T 24/24 100 29.22 0.85 2.9

M. orale CH19299T 24/24 100 31.85 1.81 5.7

M. pneumoniae FHT 24/24 100 33.03 0.73 2.2

M. salivarium PG20T 24/24 100 31.14 0.87 2.8

M. synoviae WVU 1853 T 24/24 100 33.25 0.89 2.7

S. citri R8A2T 24/24 100 32.79 1.65 5.0

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Lower Limit of Detection Assessment Using Principles of qPCR

• For efficient, linear qPCR assays, 1 Ct

difference represents a 2-fold difference in

the starting quantity of the target DNA

• The MycoSEQ™ assay is highly efficient

and linear for all species tested

• Example calculation of LLOD:

The mean Ct of M.arginini at 10 GC/PCR reaction

from the previous examples = 31

- 5 GC/PCR reaction = 32

- 2.5 GC/PCR reaction = 33

- 1.25 GC/PCR reaction= 34

- 0.6 GC/PCR reaction =35

This approach to LLOD estimation is supported by experimental

results.

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The FDA Concern About Comparability of PCR to the 28-Day Culture Based Points to Consider Mycoplasma Detection Test

• The PTC test was developed over 50 years ago as a method to detect

Mycoplasma and has been required for testing mammalian cell cultures

used for producing vaccines since 1962 and therapeutics since 1972.

• The PTC test was never thoroughly validated for Limit of Detection.

• Based on the sample volume tested, 10 mL, the assumption was made

that the Lowest Limit of Detection was 1 CFU in the 10 mL test sample,

or 0.1 CFU/mL.

• In early discussions with the FDA about validation of PCR based

Mycoplasma tests, it was felt that the E.P. guidance on validation to a

LOD of 10 CFU or GC/mL may be appropriate, but perhaps not as

sensitive as the PTC test.

• The FDA requested that the initial sponsors validating PCR as a lot

release test add comparability testing to their validation protocols.

• Comparability was assessed as follows: sets of 2 identical samples

were prepared by spiking each with a defined quantity of Mycoplasma,

1 sample is tested by PCR, the other sample is tested by the PTC test.

• Following testing of each sample, the results were compared.

23

28-day PTC Culture Test Comparability Validation Results • FDA request, comparability testing to the 28-day PTC test has been

performed as part of validation or qualification by multiple sponsors

• 3 groups have presented comparability testing results at conferences:

Amgen, Genzyme Biosurgery and Pfizer, 80 samples

at 10 CFU/mL or less

• Results:

- Positive by PCR, Positive by PTC: 45 samples

- Positive by PCR, Negative by PTC: 28 samples

- Negative by PCR, Positive by PTC: 0 samples

- Negative by PCR, Negative by PTC: 7 samples

• For the 73 samples testing positive by qPCR,38% tested negative

in the 28-day PTC test arm

• Additional Comparability Testing: Retrospective Q-PCR testing of 78

samples that had previously tested negative by PTC test

All negative by Q-PCR

- No False Positive results with the MycoSEQ™ assay

For some species qPCR achieves higher

sensitivity compared to the PTC test

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Worldwide Support Network

25

We Provide Expert, Comprehensive Support

• Training by Field Applications Scientists

• Results interpretation guidance from experts

• Instrument IQ/OQ services

• Sample prep optimization

• Validation study design

• Drug Master File (DMF) in place at FDA, Health

Canada and submitted to EMA. Enables regulatory

access to all aspects of the MycoSEQ assay design,

development and robustness testing

• Support with regulatory submissions including

attendance at meetings, Type C and Prior Approval

Supplement and support answering agency

questions

26

Legal Disclaimer

For Research Use Only. Not for use in diagnostic procedures.

• © 2015 Thermo Fisher Scientific Inc. All rights reserved. TaqMan is a registered trademark of Roche Molecular Systems,

Inc., used under permission and license. All other trademarks are the property of Thermo Fisher Scientific and its

subsidiaries.

27

Appendix

28

Advantages of Fully Integrated, Quality Controlled Kits

Enables Consistent Performance Kit to Kit, Lot to Lot, Year to Year

Minimizes

Challenging Test Samples Leverage our experience gained from solving sample preparation

challenges around the world.

• Development of SOP’s for

preparation of standards and controls

• Preparation and qualification of standards

and controls

• Internal development and optimization

investment

• Procurement and qualification of reagents

from multiple vendors

Customer Support • Field Application Scientist support

minimizes development of specialized

internal training program and enables rapid

implementation.

• Equipment validation support, protocols and execution.

• Test method validation examples and guidance.

• Trouble shooting: Experienced technical support if a

problem is encountered

29

Test Sample Types Evaluated in the Field

• High titer CHO culture from Bioreactors

• High titer NS0 culture from Bioreactors

• Cell Culture Vaccine Manufacturing

• Transgenic Milk

• Research Cell lines

• Bio-Assay cell lines

• Stem Cell Culture

• Lymphocyte proliferation culture for autologous transplantation

• Chondrocyte culture for tissue therapy

• Serum

• Cell Culture Media

• Bovine Serum Albumin

• Yeastolate

• Tryptic Soy Broth

• Bacto Casamino Acids

• Trypsin

• Cholesterol Concentrate

30

DNA

Added

M1 M2 M3 M4 M5

Un-cut Sau96I Un-cut Sau96I Un-cut Sau96I Un-cut Sau96I Un-cut Sau96I

1 ng 122% 91% 122% 100% 95% 88% 103% 93% 85% 105%

0.1 ng 95% 94% 95% 108% 109% 86% 99% 107% 82% 110%

10 pg 91% 84% 91% 98% 96% 89% 84% 98% 86% 101%

1 pg 88% 82% 88% 87% 91% 79% 85% 82% 66% 101%

0.1 pg 91% 87% 91% 80% 100% 87% 82% 86% 76% 115%

PrepSEQ™ with ResDNASEQ™ assays Sample Type Evaluation Results

Full length and

enzymatically

degraded DNA

evaluated

Matrix Buffers IgG

M1 Ion Exchange 0.8 M NaCl

20 mM NaPO4 (pH 7.5) 10 mg/ml

M2 Hydrophobic

Interaction

0.75M Ammonium sulfate

50 mM NaPO4 (pH 6)

10 mg/ml

M3 Ion Exchange 0.7 M KPO4 (pH 6) 10 mg/ml

M4 Protein A 100 mM Sodium citrate pH 3.0 10 mg/ml

M5 Purified Drug

Substance

3% Mannitol; 2% Sucrose

10 mM L-Arginine

0.01% Tween 20

100 mg/ml

Surrogate Matrices: Typical Monoclonal Antibody Purification

0.5

1

1.5 2

10

kb

Un

-cu

t

Sa

u96

I

Offers consistent recovery and quantitation of high and

low molecular weight DNA from multiple test sample matrices

31

DNA Recovery

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

Vero CHO

Spike 1

Spike 2

Spike 3

% Recovery

Vero CHO

Sample 1 7.6 7.0

Sample 2 8.8 10.8

Sample 3 7.0 10.0

Avg % Recovery 7.8 9.3

%CV 12.0 22.0

% Recovery Experimental Outline: • 20 pg of CHO or Vero cell genomic DNA spiked into

100 µl of surrogate matrix consisting of 50 mg/ml IgG

in Mannitol, Sucrose, L-Arginine and Tween-20

• DNA extracted according to column manufacturers

protocol

• DNA quantitated using resDNASEQ™ Vero or CHO

assay

• DNA recovery calculated and reported as percent

recovery

Results Using Common Spin Column Method

32

Highly efficient DNA recovery increases assay sensitivity:

PrepSEQ™ Sample Preparation Kit:

Identical, low levels of Mycoplasma bovoculi in all test samples

NO TARGET

~ 100 000 HeLa CELLS

Commercial Sample Preparation CT 37–39 NEGATIVE

PrepSEQ™ Sample Preparation Ct 35–36 POSITIVE

~ 100 000 HeLa CELLS

TARGET

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Sensitivity Comparison: Plate Culture vs. qPCR

Mycoplasma Spike;

CFU/mL Positive / Negative CT Tm (°C) Derivative

0 Negative n/a <75 <0.04

0.004 Negative n/a 77 <0.05

0.04 Negative 38 77 <0.06

0.4 Positive (Low Level) 35.5 78.7 <0.08

4 Positive 30.6 79 >0.1

~ 40 Positive 27.6 79 >0.1

• Mycoplasma arginini culture

• 10-fold dilution series of Mycoplasma culture

• Spike 13 mL samples of CHO cells (108 total cells)

• DNA purified from sample using PrepSEQ™ Sample Preparation Kit + Module M

• Analyzed by MycoSEQ™ Mycoplasma Q-PCR assay

• CFU determined by plate culture of Mycoplasma dilution series

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Broad Detection of Mycoplasma Species

Inclusion Panel (Partial)

Acholeplasma granularum Mycoplasma genitalium Mycoplasma synoviae

Acholeplasma laidlawii Mycoplasma gypis Mycoplasma testudinis

Acholeplasma pleciae Mycoplasma hominis Mycoplasma timone

Mycoplasma alkalescens Mycoplasma hyorhinis Spiroplasma citri

Mycoplasma alvi Mycoplasma imitans Spiroplasma endosymbiont

Mycoplasma anseris Mycoplasma indiense Spiroplasma insolitum

Mycoplasma arginini Mycoplasma lagogenitalium Spiroplasma kunkelii

Mycoplasma auris Mycoplasma lipofaciens Spiroplasma melliferum

Mycoplasma buccale Mycoplasma mobile Spiroplasma mirum

Mycoplasma californicum Mycoplasma molare Spiroplasma phoeniceum

Mycoplasma canadense Mycoplasma mycoides Spiroplasma poulsonii

Mycoplasma capricolum Mycoplasma neurolyticum Spiroplasma sp.

Mycoplasma caviae Mycoplasma orale Mycoplasma bovirhinis

Mycoplasma collis Mycoplasma phocidae Mycoplasma bovis

Mycoplasma cricetuli Mycoplasma pirum Mycoplasma bovigenitalium

Mycoplasma equirhinis Mycoplasma pneumoniae Mycoplasma canis

Mycoplasma fermentans Mycoplasma salivarium Mycoplasma felis

Mycoplasma gallinaceum Mycoplasma simbae Mycoplasma fastidiosum

Mycoplasma gallisepticum Mycoplasma sp. Mycoplasma muris

Mycoplasma gateae Mycoplasma spumans Mycoplasma pulmonis

• >90 species

• Related Acholeplasma laidlawii and Spiroplasma citri

• Common isolated species recommended for testing and validation (listed in red)

35

Specificity: External Validation Results

Species PCR Reaction #1 PCR Reaction #2 PCR Reaction #3

CT TM D.V. +/- CT TM D.V. +/- CT TM D.V. +/-

Hamster Und. 71.7 0.018 - 38.9049 72.1 0.047 - 39.4103 71.7 0.032 -

Human Und. 72.1 0.0094 - Und. 72.1 0.027 - Und. 72.1 0.0185 -

Mouse 39.8227 72.8 0.024 - Und. 72.8 0.016 - 38.136 72.8 0.028 -

B. cereus Und. 70.4 0.0095 - Und. 72.4 0.017 - Und. 72.8 0.021 -

B. subtilis 37.7234 75.2 0.0285 - 38.5207 74.9 0.0198 - 37.5753 75.2 0.0325 -

C. albicans Und. 72.4 0.0113 - Und. 65.5 0.0076 - Und. 72.8 0.0088 -

Cl. perfringens Und. 71.7 0.017 - Und. 72.4 0.011 - 39.6925 72.4 0.031 -

E. coli Und. 65.5 0.008 - Und. 72.1 0.0172 - Und. 72.1 0.0079 -

St. aureus Und. 65.5 0.0095 - 39.2726 73.2 0.0385 - Und. 65.5 0.009 -

St. epidermidis Und. 72.8 0.0125 - Und. 73.2 0.0123 - Und. 73.2 0.015 -

Mc. luteus 39.9058 72.8 0.0305 - 39.0225 72.1 0.0285 - Und. 72.4 0.015 -

• Closely related bacterial and common host species

• Tested 10,000 genome copies/reaction

36

Results Interpretation/Acceptance Criteria

All three criteria must be met for a test sample to be

positive for the presence of mycoplasma DNA

Cycle at Threshold (Ct)

• A measure of target DNA

level at the beginning of

the PCR reaction.

• Acceptance criteria:

- For Routine Testing:

Ct less than or equal to 36.

Derivative Value (DV)

• A measure of specific

amplicon quantity

generated during PCR

reaction

• Acceptance Criteria:

DV greater than 0.08

Melting Temperature (Tm)

• A measure of amplicon size

and base composition

(known for Mycoplasma

using this assay)

• Acceptance Criteria:

Tm between 75 and 81

degrees C

37

Comparison with First Generation Mycoplasma Assay

The original Applied

Biosystems

Mycoplasma detection

assay was based on

TaqManTM technology

For Mycoplasma detection, there are significant advantages

using SYBRTM Green detection technology

38

Mycoplasma Detection: SYBRTM Green assays vs. TaqManTM assays

The first generation Applied Biosystems Mycoplasma Detection Assay

utilized TaqManTM technology. This assay was not commercialized

Assay Design

SYBR Green

Multiplexed,

31 primers, over 90

species detected

Specificity

SYBR Green

Detects only

Mycoplasma

SYBR Green

3 parameters: Ct, Tm

and Derivative Value

SYBR Green

1 well for per test

sample, 1 well for

positive control

Test Result

Interpretation Test Set-up

TaqMan

4 separate primer/

probe sets, 80 species

detected

TaqMan

Some related bacterial

species detected

TaqMan

4 wells for per test

sample, 4 wells for

positive controls

TaqMan

1 Parameter, CT

39

Sensitivity Comparison: SYBRTM Green assays vs. TaqManTM assays

Sample Name SYBR Green assays (Ct) TaqMan assays (Ct)

Mycoplasma arginini 18.2 20.6

Mycoplasma gallisepticum 17.5 19.6

Mycoplasma orale* 23.0 24.7

Mycoplasma hyorhinis 18.4 21.2

Mycoplasma fermentans 18.3 20.8

Mycoplasma pirum 16.6 18.8

Mycoplasma pneumoniae 19.3 25.3

Mycoplasma synoviae 19.1 20.4

Mycoplasma salivarium 22.6 21.0

Mycoplasma hyopneumoniae 20.0 23.0

Acholeplasma laidlawi 18.5 18.6

Spiroplasma citri 17.8 28.5

Analysis of identical samples of purified Mycoplasma DNA (~1ng/rxn)

• Lower Ct correlates with increased sensitivity

• For most common species, SYBR Green offers significant sensitivity advantages

40

From Mycoplasma Screening to Identification

Screening Is Mycoplasma present?

Identification What species?

Compatibility with MicroSEQTM Microbial Identification System

MycoSEQ™

Mycoplasma

Detection System

MicroSEQTM Microbial

Identification System

MicroSEQTM 500 bp Library v2013

41

Mycoplasma Detection and Identification:

Ct = 22.4

Tm = 77.5oC

D.V. ~ 0.281

MycoSEQ ™ data

Reserve of PrepSEQ eluate analyzed with MicroSEQTM 500 Bacterial Identification kit:

MycoSEQ ™ Result: Ct = 22.4, Tm = 77.5, D.V. = 0.281, Positive for Mycoplasma

Example from Customer Testing