Mycoplasma gene detection kit

March 31, 2016

Myco Finder
Validation Data

Shimadzu Diagnostics CorporationCopyright © Shimadzu Diagnostics Corporation All rights reserved.

11. Overview
Validation data was obtained in reference to the following information in order to validate the performance of MycoFinder, a mycoplasma gene detection kit.
1. Japanese Pharmacopoeia 17th Edition (March 7, 2016 The MHLW Ministerial Notification No. 64)
Reference information p2395-2399“Negative Tests for Mycoplasma of Cell Substrate for the Production of Medicines Applied with Biotechnology
and Medicines Originated in Biology”
2. PHARM TECH JAPAN (2016) vol.32, No.1, 93-94

2. Myco Finder validation items and test conditions
Table 1 shows reagents used for extracting DNAs and conditions of the real-time PCR test, and Table 2 shows
information about mycoplasma reference strains used for validation.
Table 1. Myco Finder validation detection condition
DNA extraction reagent QIAGEN’s DNeasy Blood and Tissue Kit
BioRad’s CFX96
PCR system
95°C, 10 seconds
98°C, 3 seconds
45cyclesPCR condition
60°C, 1 second

Fluorescence detection wavelength: FAM + ROX (or FAM + HEX)
Performed using the software that comes with PCR system (CFX
Judgment ofmeasurement result

Manager™ Software). Analysis method: Regression method,Baseline Subtracted Curve Fit mode

*Copies/mL= [DNA contents (µg/mL)] x 106 x (0.978 x 109)/[genome size]

Table 2. Mycoplasma reference strains used for validation
Reference strains produced under the supervision of Mr. Norio Shimiu, Assistant Professor of Tokyo Medical and Dental University Center for
Stem Cell and Regenerative Medicine were used for validation.

Strain nameGC (copies/mL)*

CFU (CFU/mL)

GC/CFUM. pneumoniae

6.0E+08

2.9E+07

20.7

M. hyorhinis

1.4E+09

4.2E+08

3.4

A. laidlawii

3.9E+09

3.5E+08

11.2

M. fermentans

1.3E+10

3.3E+09

3.9

M. arginini

4.1E+09

2.2E+09

1.8M orale

1.3E+108.3E+08

16.3

M salivarium

4.2E+09

2.5E+09

1.7

Copyright © Shimadzu Diagnostics Corporation All rights reserved.

2

3. Specificity
Using the DNAs extracted from the 7 reference strains listed in the Table 2 as templates, PCR was performed with
Myco Finder’s primer, and then sequence analysis was performed. The result showed that Myco Finder’s primer can
specifically amplify the 7 Mycoplasma reference strains (Table 3).
Table 3. Sequence analysis result
Strain name
Homology

A. laidlawii

100%

M. arginini

99%*

M. fermentans

99%*

M. pneumoniae

100%M orale

99%*

M. hyorhinis

99%*

M. salivarium

99%**The 1% difference derives from the base that was “undetectable (N)” in the sequence analysis.

4. Detection sensitivity
8 sets of serial dilutions for each type of test strain on Table 2 were made to yield 1, 10, and 100 CFU/mL, and acell suspension of CHO-DG44 cells at density of 1x106 cells/mL was seeded in each bacterial suspension to prepare
samples for the sensitivity test. Using these samples, sensitivity test was performed three times (each on different days)for each type of test strain to perform 24 tests in total. The only negative sample was the cell suspension mentioned
above (1x106 cells/mL).
Result of the sensitivity test is shown in Table 4 through Table 10. For all strains, 10 CFU/mL was detectable witha probability of more than 95%. This shows that Myco Finder is able to detect 10 CFU/mL, having a sensitivity level
required of an alternative to culture method.
Table 4. Sensitivity Test Result (M. orale)

Table 5. Sensitivity Test Result (M pneumoniae)

Spike (CFU/mL)

Run 1

Run 2

Run 3

Total

Spike (CFU/mL)

Run 1

Run 2

Run 3

1008/8*

8/8

8/8

24/24

100

8/8*

8/8

8/8

24/24

108/8
8/8

7/8
4/8

8/8
7/8

23/24
19/24

10

8/8
6/8

8/8
5/8

8/8
6/8

24/24
17/24

1

*Number of detections/Number of tests

1

Total

*Number of detections/Number of tests

Copyright © Shimadzu Diagnostics Corporation All rights reserved.

3

Table 7. Sensitivity Test Result (M fermentans)

Table 6. Sensitivity Test Result (M arginini)
Spike (CFU/mL)

Run 1

Run 2

Run 3

1008/8*

8/8

8/8

10

8/8
4/8

8/8
6/8

7/8
7/8

1

Spike (CFU/mL)

Run 1

Run 2

Run 3

Total

24/24

1008/8*

8/8

8/8

24/24

23/24
17/24

108/8
7/8

8/8
7/8

7/8
4/7

23/24
18/24

Total

1

*Number of detections/Number of tests*Number of detections/Number of tests

Table 8. Sensitivity Test Result (M. hyorhiniS)

Table 9. Sensitivity Test Result (M. salivarium

Spike (CFU/mL)

Run 1

Run 2

Run 3

Total

100

8/8*

8/8

8/8

24/24

108/8
5/8

8/8
5/8

8/8
3/8

24/24
13/24

1

*Number of detections/Number of tests

Spike (CFU/mL)

Run 1

Run 2

Run 3

Total

100

8/8*

8/8

8/8

24/24

108/8

7/8

8/8

23/24

16/8

4/8

3/8

13/24

*Number of detections/Number of tests

Table 10. Sensitivity Test Result (A. laidlawii)
Spike (CFU/mL)

Run 1

Run 2

Run 3

Total

100

8/8*

8/8

8/8

24/24

108/8
6/87/8
6/8

8/8
8/8

23/24
20/24

1

*Detected number/Number of tests

5. Robustness
Myco Finder’s performance was evaluated by increasing/decreasing the amount of samples from the defined amount (25µL). As
a result, it was found that detection result was not affected when the amount of sample was within 25µL ±5%.

Table 11. Effect of increased/decreased sample amount to positive ratio
Percent change*
Strain name-20%

-10%-5%0%+5%+10%+20%

M. pneumoniae

3/3**

3/3

3/3

3/3

3/3

3/3

3/3

M. hyorhinis

0/3

2/33/3

3/3

3/3

3/3

2/3

A. laidlawii

3/3

3/3

3/3

3/3

3/3

3/3

3/3

M fermentans

3/3

3/3

3/3

3/3

3/3

2/3

3/3

M. arginini

3/3

3/3

3/3

3/3

3/3

3/3

3/3

M. orale
M salivarium

1/3
1/3

2/3
2/3

3/3
3/3

3/3
3/3

3/3
3/3

2/3
2/3

0/3
0/3

* The amount of sample was increased/decreased from 25µL as a standard.
** Detected number/Number of tests

Copyright © Shimadzu Diagnostics Corporation All rights reserved.

4

6. Equivalence test ~Culture method~
In parallel with the detection test of 10 CFU/mL of the 7 strains shown in Table 2, detection test of the same sample(10 CFU/mL) by culture method was performed. As a result, the same sample was also detectable by culture method.
This shows that Myco Finder meets the condition (10 CFU/mL) required of an alternative to culture method.

Cultured for 14 days
0.2 mL
10 mL

Agar plate (N=2)

Cultured for 14 days Agar plate (N=2)

Agar plate (N=2) Agar plate (N=2)②Day 0③④0.2 mLDay 3Sample: Cell culture medium

Day 70.2 mL 0.2 mLDay 14

Liquid medium (100 mL)

Figure 1. Procedure of culture method
1. For each of the seven Mycoplasma strains, prepare a sample that contains reference strains at 10 CFU/1mL of cellsuspension.
2. Inoculate the agar plate with 0.2 ml of each sample, and culture at 37°C for 14 days ((1)).
3. Inoculate 100 mL of Hayflick (Millipore) liquid medium with 10 mL of each sample, and culture at 37°C. Take 0.2 mL ofeach culture solution on day 3, 7 and 14, and inoculate the agar plate with it, and then culture at 37°C for 14 days ((2)~(4)).
4. Confirm the existence of Mycoplasma colonies at the processes (1) through (4) above.

Table 12 Result of culture method
Length of incubation
Strain name①Day 0②Day 3③Day 7④Day 14M. pneumoniae

一一

M. hyorhinis

一一一一

M. salivarium

一++++++++++

M orale+++++++++++

A. laidlawii
M fermentans
M. arginini＋:Positive, －:Negative
When more than 1 colonies were confirmed on plate agar within the culture period, we judged it positive.
The result of M. arginine and M. orale after day 7 were negative, but this is because all the grown strains had died by then because incubation
was performed too long.

Copyright © Shimadzu Diagnostics Corporation All rights reserved.

7. Reference information~Cross-reactivity~
Although this is not listed in the Japanese Pharmacopoeia 17th Edition as validation method, cross-reactivity to the genomic
DNAs of the 25 types of bacteria, 8 types of funguses, and 3 types of mammalian cells listed in the Table 13 was also tested. Allgenomic DNAs were added to the sample so that concentration will be 1ng/reaction. The result showed that no cross-reaction wasobserved against any of the genomic DNAs.
Table 13. List of strains for which false-positive reaction was tested
25 types of bacteria

8 types of funguses

Bacteroides vulgatus

3 types of mammalian cells

Raji Cell (human)

Bacillus subtilis

Cryptococcus neoformans Candida

Mouse T lymphocyte (mouse)

Clostridium acetobutylicum

albicans

CHO cell (hamster)

Clostridium kluyveri

Mucor circinelloides

Clostridium sporogenes

Cunninghamella echinulata

Escherichia coli

Rhizomucor pusillus Absidia

Enterococcus faecalis

corynbifera Scedosporium

Gluconacetobacter xylinus

prolificans Pneumocystis carinii

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 aeruginosaEnd of document
For more information including actual values of validation data, please contact sales rep of Shimadzu Diagnostics Corporationor customer support (TEL: 03-5846-5707).

Copyright © Shimadzu Diagnostics Corporation All rights reserved.