1-1. Why mycoplasma testing is necessary?
Due to the changes in the laws (Act on Securing Quality, Efficacy and Safety of Products Including Pharmaceuticals and Medical Devices,
Act on the Safety of Regenerative Medicine Products Safety, etc.) in 2012, commercial application of regenerative medicine products is now
allowed. In line with this, mycoplasma testing has become mandatory as part of the tests to secure safety of regenerative medicine products.

<Safety test of regenerative medicine products and bio-pharmaceuticals>○Sterility Test
○Bacterial Endotoxins Test
○Virus Test
○Mycoplasma Testing

Bones

BV
Bladder

Periodontal
Eyes

Cartilage
MuscleNerve
TendonMesenchymal
stem cell
Skin
Liver
Heart muscle

Embryonicstem-cell
Pancreas

Material
Cell<Issues related to safety test>

Biologically active agent

Source: blog.goo.ne.jp/

・It is hard to secure sufficient amount of final products ofregenerative medicine as test samples because there arelimited amount of final products.
・As regenerative medicine products need to be usedsoon after production, rapid test method is required.

Japanese Pharmacopoeia 17th Edition:
Nucleic Acid Amplification Technique wasadded as a rapid mycoplasma testing
methodCopyright © Shimadzu Diagnostics Corporation All rights reserved.

Confidential11-2. What is mycoplasma?
Mycoplasma, Ureaplasma, Spiroplasma, Acholeplasma, and other bacteria that belong to Mollicutes.
<Characteristics>○Widely distributed in the animal and plant kingdoms and infection shows a
species-specificity.
○Infect humans through researchers who perform cell culture or serum used forcell culture, etc.
○Many have resistance to antibiotics.
○Pass through the filters for sterilization (0.22µm) because of the small size.
○Infected cells do not exhibit cytotoxic effect.
⇒It is hard to detect mycoplasma contamination

Fibroblast infected by Mycoplasma

EXCLI J. 2014; 13: 300–322

It is reported that cultured cells in many labs are often contaminated by mycoplasma.
⇒Development of medical products using contaminated cells will result in severe
consequences.
・Cell culture substrates used fordevelopment of medical products
・Cell/tissue-based medical products

A proper mycoplasma testing has to be performed.

Copyright © Shimadzu Diagnostics Corporation All rights reserved.

Confidential21-4. The 7 types of strains to be detected under Japanese Pharmacopoeia 17th Edition

7 types of strains were selected from the following perspectives as mycoplasma strains that may
contaminate cell substrates used for development of medical products in Japan.
・Frequency of contamination and phylogenetic point of view

・Animal-derived components used in cell culture

菌名
Strainname

Mycoplasma hyorhinis
Mycoplasma orale
Mycoplasma pneumoniae
Mycoplasma salivarium
Acholeplasma laidlawii
Mycoplasma fermentans
Mycoplasma arginini

自然宿主
Natural host
Pig
ブタ
Human
ヒト
Human
ヒト
Human
ヒト
Cow
ウシ
Human
ヒト
Cow / Goat
ウシ・ヤギ

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Confidential31-5. Method A: Culture method<Test method>

Inoculate medium (liquid medium, agar medium) with the sample and culture to detect colonies specific to mycoplasma.
MediumAmount ofsampleNo. of tests per sample

Length of incubation

Liquid medium(100mL/medium)10mL or more

1 or more culture bottles

For 14 days

Agar medium

0.2mL

0.2mL

⇒Take 0.2mL on day 3, 7, and 14, and inoculate agar plateswith them (N=2), and then incubate for 14 days.
2 or more agar plates

For 14 days
Incubate for 14 days

Agar medium (N=2)

Incubate for 14 days
Agar medium(N=2)

Agar medium(N=2)

Agar medium(N=2)

10mL
0.2mL
Sample: Cell culture medium

Day 3

0.2mL
Day 7

0.2mL
Day 14

Liquid medium (100mL)

Copyright © Shimadzu Diagnostics Corporation All rights reserved.

It takes 28 days from thestart of culture to the end of
the test
Confidential41-6. Method B: DNA staining method<Test method>

This method indirectly detects mycoplasma that have grown in cell culture by inoculating the indicator cells (Vero cells) with the test sample
and staining the mycoplasma DNA with fluorescent staining dye to observe small extra-nuclear fluorescence spots.

Preparation of indicator cells
is necessary

Incubate indicator cells (Vero cells) in culture dish for one day(Use 2 or more dishes per sample)
Add 0.5 mL of test sample (culture supernatant) and incubate for 3 to 6
days
Fix the cells, stain them with benzimidazole fluorescent stain, and observeusing microscope (x 400-600)

Test takes 4 to 7 days
Positive

The result is positive when 5 or more cells out of 1000 cells are observed tohave small extra-nuclear fluorescence spots.

Experience and skills are necessary tojudge positive or negative.

Negative

Source: Jikken Recipe by Sigma-aldrich

Judgement of the result is difficult as this is not amethod specifically for mycoplasma.

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Confidential51-7. Method C: Nucleic Acid Amplification Technique (NAT methods)<Test method>
Extract DNAs from the sample and amplify them using mycoplasma-specific primers to detect mycoplasma.
Under Japanese Pharmacopoeia 17th Edition…
・Validation methods for NAT technique are presented⇒As far as validation is performed, various methods can be used
・Can be used as an alternative to Method A and Method B⇒If sufficient sensitivity is confirmed through proper validation, the method can be used as an alternative to Method A and
Method B
Alternative to Method A: 10CFU/mL is detectable for all 7 mycoplasma strains
Alternative to Method B: 100CFU/mL is detectable for all 7 mycoplasma strains.

Nucleic Acid Amplification Technique can be used as a rapid mycoplasma testing method thathas superior sensitivity and specificity, as far as proper validation is performed.
Copyright © Shimadzu Diagnostics Corporation All rights reserved.

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2-2. Product outline (Packaging specification)
Paid samples of Prevrsion (product code: 69201), a mycoplasma gene detection kit, are on sale
We have agreed on delivery specification of Preversion with the manufacture (NTS) (The manufacturer performs manufacturing,packaging, and labeling of the kit).
1 kit contains 48 tests, can be stored in a cool place (2-8℃). Valid for 6 months from the date placed on the market (ultimate goal is 12
months).
<Package presentation>
Mycoplasma gene detection kit Preversion (48 tests)[Appearance](1)(2)[Constituent reagents](1) Test strip A (white) x 6(2) Test strip B (transparent) x 6(3) Positive control (2 x 10^3copies/μL) x 1(4) Negative control (DNase Free Water) x 1(5) Flat cap x 6
Copyright © Shimadzu Diagnostics Corporation All rights reserved.

(3)

(4)

(5)
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2-3. Test method outline
Following is a rough outline of mycoplasma testing using this kit

Sample: cell culture⇒Cell substrate used for the development of
medical products
Cell/tissue-based medical products, etc.

Judgment (can be automatically judged using
analysis software)

Centrifugal
concentration

Real-time PCR
Copyright © Shimadzu Diagnostics Corporation All rights reserved.

Extraction of nucleic acid
⇒The kit by QIAGEN is recommended

Reagent preparation (detection)⇒Mycoplasma gene detection kit
Confidential8

2-4. Real-time PCRThis kit detects mycoplasma nucleic acid by real-time PCR
TaqMan probe methodProbe1) Thermal denaturation
PrimerFluorescentmaterial

Quencher

2) Primer annealing / probe hybridization
Polymerase

Positive

Hybridize3) Elongation

Negative

Fluorescent signal
Source: TAKARA website

Probes are degraded during PCR and a fluorescent signal is emitted.
By detecting the fluorescent signal, amplification of nucleic acid can be
captured.
Copyright © Shimadzu Diagnostics Corporation All rights reserved.

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92-5. How to use the kit (reagent preparation)
Preparation of reagent is not necessary because dried reaction reagent is set in the reaction tube for each testAs a potent amplification enzyme is used, detection can complete within one hour from the start of amplification<Usage method (example)>(1) Add 25μL of the DNA detection sample at the right concentration to the test strip B(2) After dissolving the solid reagent in the test strip B by pipetting, move all to the test strip A(3) After dissolving the solid reagent in the test strip A by pipetting, attach the flat cap(4) Detect by real-time PCR system
SampleTest strip B(1)

Attach flat cap

Test strip A(2)

The operation completes in 4 steps!
Can choose from white and transparent strips for detection!
Depending on the kind of real-time PCR system used, the sample can be added to either
test strip A or test strip B.

Copyright © Shimadzu Diagnostics Corporation All rights reserved.

(3)

(4)
Detect by real-time PCRsystemConfidential10

2-6. Product feature ~ Special positive control~False-positive result due to contamination of negative sample by positive control is identifiable<Example of detection of positive control>
Amplification curve of mycoplasma fermentans (10cfu/mL)

Amplification curve of positive control (1000copies/reaction)

Fluorescent signal: FAM(blue), HEX(green),
ROX(yellow)
Fluorescent signal is positive only for FAM

Fluorescence: FAM(blue), HEX(green),
ROX(yellow)
Fluorescent signal is positive for FAM, HEX,
and ROX<Design of positive control>
Conventional positive control
Mycoplasma-specific primer

This kit’s positive control

Mycoplasma-specific probe

Mycoplasma-specific primer

Positive control-specific probe

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2-7. Comparison between commercially available kits ~basic specification~
Product nameMycoFinder (newly marketed kit)

MycoTOOL Mycoplasma real-time PCR Kit

MycoSEQ Mycoplasma Real-Time PCR

Sold byJP compliance
Principle
No. of tests / kit

Shimadzu Diagnostics Corporation

Roche

ABI

Compliance with JP17 is intended
Real-time PCR method
48

Compliance with EP is intended
Real-time PCR method
About 160

Compliance with JP 17 is intended
Real-time PCR (SYBR) method
100

Reagent composition-Strip A: 6 strips-Strip B: 6 strips
・Positive Control: 8vial
・Water: 8vial

・Recovery Control: 1vial(200μL)*PCR reaction reagent is solidified in the strip

・PCR Master: 5vial
・UNG: 1vial (180μL)
・PCR Enhancer: 1vial (180μL)
・Detection Mix(Probe): 1vial (220μL)
・Detection Mix Recovery Control(Probe):1vial (140μL)
・Positive Control: 1vial (800μL)
・Water: 2vial (1mL)

・Primer Mix: 1tube(325μL)・Negative Control: 1tube(1000μL)
・SYBR Master mix: 2tube(1000μL)
・DNA Control: 1tube(400μL)*Water is not included

Sensitivity

10cfu/ml

10cfu/ml

1-10copies/tube

Specificity

140 species

90 species

UNG system

None

Internal control

Theoretically 142 species
Identification of positive control against test
sample is possible
None

GAPDH

None

Storage temperature

Cool place (2-8℃)-20℃-15～-25

Time

2 hours (in case of a potent PCR)

5 hours

Unknown

Measurement device

Short type only
(White and transparent are available)

Universal

Universal

Contamination measure

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2-8. Use example of the kit
This kit can detect mycoplasma in cell culture ⇒ It can be used not only for the testing of final products,
but also for the voluntary inspection during culture.

CultureCell culture starts

Take a sample from culture solution

Voluntary inspection⇒Monitor the amount of mycoplasma in
culture solution

Copyright © Shimadzu Diagnostics Corporation All rights reserved.

Products such as cells

Mycoplasma testing(Compliant with JP17)*To implement PCR, pre-validationis necessary.
Confidential133. Performance evaluation of the kit andvalidation to comply with JP

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3-1. Why validation is necessary?
Do users have to perform full-validation at their own facilities?
⇒The revision proposal states, “when a commercially available kit is used for NAT testing, the full validation data fromthe manufacturer can be used as an alternative to the validation data by the user.”If the maker’s validation data is available, the user does not have to perform full-validation

But!
The differences in cell types of the sample, extraction method, detection device, etc. may affect thesensitivity and reproducibility. Therefore a performance validation need to be performed at the user facility.
Users need to confirm that the same validation result as the one presented by the maker can beobtained at their own facilities.
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3-2. Method C (NAT method) validation outline
The JP17 requires a proper validation be performed.
<Outline of validation method>(1) Validation of NAT method
 Specificity: Detection of 7 species
 Detection sensitivity
Mycoplasma reference strains for which characteristic analysis has been performed and concentration (CFU or
number of copies, etc.) has been clarified or strains authorized by the international standards have to be used.
Sensitivity for detecting mycoplasma contained in cell suspension has to be evaluated.
The test has to detect mycoplasma with a probability of more than 95%.
 Robustness of the analysis method
The method has to be proved reliable under normal use.
(2) When NAT method is used as an alternative to Method A or Method B Equivalence test
Alternative to Method A: 10CFU/mL is detectable for all 7 strains of mycoplasma
Alternative to Method B: 100CFU/mL is detectable for all 7 strains of mycoplasma
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3-3. Validation implementation plan
Details of kit validation is as follows.
<Details of validation>
Item

ResultSpecificity

Pass

Revised JP

Detection
sensitivity
Robustness

Pass

Detail
It has to be confirmed that nucleic acids derived from the 7 reference strains for which sequence analysis has
been performed are detectable.
1, 10, and 100CFU/mL have to be tested to confirm that 10CFU/mL is detectable with a probability of 95% or more.

Underevaluation Tolerance range has to be evaluated by increasing/decreasing the sample amount from the defined 25uL.

Equivalence (1)Pass

It has to be confirmed that the seven strains are detectable at 10CFU/mL .

Equivalence (2)(culture method)Pass

It has to be confirmed that the samples used for sensitivity test (10CFU/mL) are also detectable by culture method.

Additional
data

Deviceevaluation
Safety test

Underevaluation Shimadzu Corporation device is evaluated.
Underevaluation Safety test for 6 month is to be performed in March (Up to 4 month data has been obtained.)
Copyright © Shimadzu Diagnostics Corporation All rights reserved.

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3-4. Validation conditions
Conditions of the kit validation are as follows.
<Materials and devices used>

DNA extraction kit:
DNeasy Blood & Tissue Kit(QIAGEN)
Real-time PCR system: CFX96(Bio-Rad)
Cell:
CHO-DG44 cells (1 x 10^6 cells/mL)
Mycoplasma reference strains: see below<Reference strain information> GC values and CFU values of the reference strains prepared in our facility used for evaluation are shown below. GC
values and CFU values were measured in accordance with the National Institute of Health Sciences method*.
Strain name

OriginM. pneumonieae
M. hyorhinis

NBRC14401
NBRC14858

A. laidlawii

NBRC14400

M. fermentans
M. arginini
M. oraleM. salivarium

NBRC14854
ATCC23838
NBRC14477
NBRC14478

No. of genomic
copies（copies/mL）

CFU(CFU/mL)

GC/CFU

6.0E+08
1.4E+09
3.9E+09
1.3E+10
4.1E+09
1.3E+10
4.2E+09

2.9E+07
4.2E+08
3.5E+08
3.3E+09
2.2E+09
8.3E+08
2.5E+09

20.7
3.4
11.2
3.9
1.8
16.3
1.7

*Health Labour Sciences Research Grant 2014 Research Report: Evaluation of the safety of innovative drugs against
viruses and infectious agents
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3-5. Specificity test
To confirm that 7 types of test strains of mycoplasma are specifically detected, following two experiments were conducted.
Performed PCR using the genomic DNAs from the 7 reference strains as templates
Performed sequence analysis of the PCR product
The DNA sequence of the PCR product has more than 99%
equivalence with mycoplasma gene it drives from.
Performed sensitivity test using the DNA from the sample(cell suspension + mycoplasma reference strains)

10CFU/mL was detected for the 7 strains.

It was confirmed that the kit’s primer canamplify mycoplasma gene.

Mycoplasma was specifically detected even when cellderived DNAs exist.

It was confirmed that this kit can specifically detect the 7 mycoplasma species.
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3-6. Sensitivity test (Equivalence test)
Here is the result of the sensitivity test using theserial dilution.
Sensitivity is to be calculated by statistical analysis.
Detection limit for M.orale

Detection limit for M.arginini
Spike(cfu/mL)

Run 1

Run 2

Run 3

Total

100

8/8

8/8

8/8

24/24

10

8/8

8/8

7/8

23/24

1

4/8

6/8

7/8

17/24

Detection limit for M.hyorhinis

Spike(cfu/mL)

Run 1

Run 2

Run 3

Total

Spike(cfu/mL)

Run 1

Run 2

Run 3

Total

100

8/8

8/8

8/8

24/24

100

8/8

8/8

8/8

24/24

10

8/8

7/8

8/8

23/24

10

8/8

8/8

8/8

24/24

1

8/8

4/8

7/8

19/24

1

5/8

5/8

3/8

13/24

Detection limit for M.pneumoniae

Detection limit for M.salivarium

Spike(cfu/mL)

Run 1

Run 2

Run 3

Total

Spike(cfu/mL)

Run 1

Run 2

Run 3

Total

100

8/8

8/8

8/8

24/24

100

8/8

8/8

8/8

24/24

10

8/8

8/8

8/8

24/24

10

8/8

7/8

8/8

23/24

1

6/8

5/8

6/8

17/24

1

6/8

4/8

3/8

13/24

Detection limit for M.fermentans

Detection limit for A.laidlawii

Spike(cfu/mL)

Run 1

Run 2

Run 3

Total

Spike(cfu/mL)

Run 1

Run 2

Run 3

Total

100

8/8

8/8

8/8

24/24

100

8/8

8/8

8/8

24/24

10

8/8

8/8

7/8

23/24

10

8/8

7/8

8/8

23/24

1

7/8

7/8

4/7

18/24

1

6/8

6/8

8/8

20/24

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3-7. Equivalence test (NAT technique)
The sensitivity test result showed that the 7 test strains specified by JP17 are detectable at 10-CFU/mL with a probability of 95% or
more.

⇒The method meets conditions required of an alternative to Method A (culture method)
1st test

2nd test

3rd test

Total

Detection rate

8/8
8/8
8/8
8/8
7/8
8/8

7/8
7/8
8/8
8/8
8/8
8/8

23/24

95.8%

23/24

95.8%

24/24

100.0%

24/24

100.0%

23/24

95.8%

M. salivarium

8/8
8/8
8/8
8/8
8/8
7/8

23/24

95.8%

A. laidlawii

8/8

7/8

8/8

23/24

95.8%

SampleM. arginini
M. fermentans
M. hyorhinis
M. pneumoniae
M. orale

Negative sample

Reference strains (10 CFU)
+ cell suspension

Cell suspension

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3-8. Equivalence test (culture method)
Using the same sample as the sensitivity test (cell suspension + reference strains), detection test was performedby culture method.
Incubated for 14 days
0.2mL

Agar medium (N=2)①

Incubated for 14
days

10mL
Sample: Cell culture medium

Agar medium (N=2)

0.2mL

Agar medium(N=2)

②

Day 3

Agar medium (N=2)

③

④

0.2mL

Day 7

0.2mL
Day 14

Liquid medium (100mL)

Plate medium (sampling from liquid medium)
平板培地(液体培地からのサンプリング)
Day 7 (3)Strainname
Day 14 (4)Day 1
medium 開始日①
Day 3 (2)Inoculated
amount Liquid液培
菌名
接種量
３日目②
７日目③
14日目④
M.pneumoniae
ww＋++
M.hyorhinis
＋
+
+
+
+
A.laidlawii
＋
+
+
+
+
10CFU/mL
M.fermentans (100CFU/ボトル）
＋
+
+
+
+
(100CFU/bottle)
M.arginini＋++
M.orale＋++M.salivarium＋+++

It was confirmed that all 7 sample strains yield positive result at 10 CFU/mL.
⇒This shows that the sensitivity test and the culture method have the same sensitivity.
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3-9. Additional data ~Cross reactivity test~
No cross-reactivity was observed for 25 types of bacteria, 8 types of funguses, and 3 types of mammalian cells.
<Cross-reactivity>
25 types of bacteria
Bacteroides vulgatus
Bacillus subtilis
Clostridium acetobutylicum
Clostridium kluyveri
Clostridium sporogenes
Escherichia coli
Enterococcus faecalis
Gluconacetobacter xylinus
Klebsiella pneumoniae
Lactobacillus acidophilus
Lactobacillus bulgaricus
Lactobacillus casei

Propionibacterium acnes
Sallmonella enterica
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus mutans
Streptococcus pneumoniae
Streptococcus bovis
Streptococcus avermitilis
Rhodococcus erythropolis
Rothia dentocariosa
Tetragenococcus halophilus
Kocuria rhizophila
Pseudomonas aeruginosa

9 types of funguses
Cryptococcus neoformans
Candida albicans
Mucor circinelloides
Cunninghamella echinulata
Rhizomucor pusillus
Absidia corynbifera
Scedosporium prolificans
Pneumocystis carinii

3 types of mammalian cells
Raji Cell (human)
Mouse T lymphocyte (mouse)
CHO cell (hamster)The genome used for the test is 1ng/reaction**Equivalent to about 2 x 10^5copies assuming the average genome size of the bacteria is 5 x 10^6bp
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