February 27, 2017

Myco Finder Validation Data

Shimadzu Diagnostics Corporation
Research Department

1.

Overview
To evaluate the performance of the mycoplasma gene detection kit (Myco Finder), validation data was obtained with reference to thefollowing two information.

1. The Japanese Pharmacopoeia,17th Edition (Ministry of Health, Labour and Welfare Notification No. 64, March 7, 2016)
Reference Information
Mycoplasma negative test for cell substrates used in the manufacture of biotechnology-applied pharmaceuticals/biologicallyderived pharmaceuticals p 2395 -2399

2. PHARM TECH JAPAN (2016) vol. 32, No. 1, 93 -942.

Details of Myco Finder Validation and Experimental Conditions
The experimental conditions performed with the real-time PCR devices are shown in Table 1, and the mycoplasma information of thisvalidation is shown in Table 2.

Table 1. Myco Finder validation detection criteria
DNA extraction reagent QIAGEN: QIAamp UCP DNA Micro Kit
PCR device

BioRad: CFX 96

PCR conditions

95 °C, 10 seconds
98℃, 3 seconds}45 cycles
60℃, 1 second
Fluorescence detection wavelength: FAM + ROX (or FAM + HEX)

Judgment ofmeasurement results

Analysis performed using the software provided with the PCR device (CFX ManagerTM software):Regression method, Baseline Subtracted Curve Fit mode

Table 2. Mycoplasma reference material used for validation
For validation, the reference materials prepared under Associate Professor Norio Shimizu of the Tokyo Medical and Dental University Research Center for
Regenerative Medicine were used.

Species nameGC (copies/mL) *A. laidlawii
M. arginini
M. fermentans

CFU (CFU/mL)

GC/CFU

3.5 x10811.2

2.2 x109

1.8

3.3 x109

3.9

1.4 x 109

4.2 x 108

3.4

8.3 x10816.3

2.9 x107

20.7

2.5 x 109

1.7

3.9 x

109

4.1 x

109

1.3 x

M. hyorhinis

1010

1.3 x

1010

M. pneumoniae

6.0 x

108

M. salivarium

4.2 x 109

M. orale*Copies/mL = [DNA contents (μg/mL)] x

106x (0.978 x109)/[genome size]

3. Specificity
3-1.Specific amplification of target region
PCR using primers used in the Myco Finder was performed using DNA extracted from the reference materials of the seven species shownin Table 2 as a template, followed by sequence analysis. The results showed that the primers of Myco Finder could specifically amplify theseven mycoplasma species (Table 3).

2Species name

Homology

A. laidlawii
M. arginini
M. fermentans
M. hyorhinis
M. oraleM pneumoniae
M salivarium

100%
99%*
99%*

99%*
99%*
100%
99%*

•Include & Undetectable

3-2.Cross-reactivity with other species
Although not included in the validation method specified in the Japanese Pharmacopoeia,17th Edition, the cross-reactivity of the genomic
DNA of the 25 bacterial species, eight fungal species, and three mammalian cell species shown in Table 4 was evaluated. Genomic DNA wasadded to achieve at least 1 ng/reaction in each case. As a result of the evaluation, no cross-reactivity was observed for any of the genomic
DNAs listed in Table 4.

Table 4. List of species tested for cross-reactivity
25 bacterial species
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
Salmonella enterica
Staphylococcus aureusStaphylococcus epidermidis
Streptococcus mutans
Streptococcus pneumoniae
Streptococcus bovis
Streptococcus avermitilis
Rothia dentocariosa
Tetragenococcus halophilus
Kocuria rhizophila
Pseudomonas aeruginosa8 fungal species
Cryptococcus neoformans
Candida albicans
Mucor circinelloides
Cunninghamella echinulata
Rhizomucor pusillus
Absidia corynbifera
Scedosporium proficans

Pneumocystis carinii

33 mammalian cell species
Raji Cell (Human)
Mouse T lymphocyte (Mouse)
CHO cell (Chinese hamster)4. Detection Sensitivity
For the seven bacterial species to be detected shown in Table 2, eight different dilution series of 1, 10, and 100 CFU/mL wereprepared, and a cell suspension of CHO cell DG 44 strain 1X106 cells/mL was added to each of them to serve as a sensitivity test sample.
These were used for performing three tests on separate days, for 24 tests in total. Only the above cell suspensions (1X106 cells/mL) wereused as negative samples.
The results of the detection sensitivity test are shown in Tables 5 to 11. For all species, 10 CFU/mL was detectable with a probability ofmore than 95%. This indicates that Myco Finder has the sensitivity to meet the requirement of 10 CFU/mL, as an alternative to culture.

Table 5. Sensitivity test results (A. laidlawii)
Spike(CFU/mL)

Run
1

2100
8/8
8/8
108/8
8/8
16/8
7/8
3/3 means detected 3 times out of 3 tests.

38/8
8/8
8/8

Table 6. Sensitivity test results (M. arginini)

Total

Positive rate
(%)

24/24
24/24
21/24

100.0
100.0
87.5

Table 7. Sensitivity test results (M.fermentans)
Spike(CFU/mL)

Run
12100
8/8
8/8
108/88/81
3/8
5/8
3/3 means detected 3 times out of 3 tests.

38/8
8/8
7/8

Spike(CFU/mL)

12100
8/8
8/8
10
8/8
8/8
1
4/8
4/8
3/3 means detected 3 times out of 3 tests.

3
8/8
8/8
4/8

Run
1
2100
8/8
8/8
10
7/8
8/8
1
5/8
3/8
3/3 means detected 3 times out of 3 tests.

38/8
8/8
2/8

2

100
8/8
8/8
10
8/8
8/8
1
4/8
5/8
3/3 means detected 3 times out of 3 tests.

Total
24/24
24/24
15/24

100.0
100.0
62.5

TotalPositive rate
(%)

24/24
24/24
12/24

100.0
100.0
50.0

Spike(CFU/mL)

Positiverate (%)

24/24
24/24
13/24

100.0
100.0
54.2

38/8
8/8
4/8Run121008/8
8/8
108/8
8/8
1
1/8
3/8
3/3 means detected 3 times out of 3 tests.

3
8/8
8/8
4/8

Total

Positiverate (%)

24/24
24/24
8/24

100.0
100.0
33.3

Table 10. Sensitivity test results (M. pneumoniae)
Spike(CFU/mL)

Total

Positive rate(%)

24/24
23/24
10/24

100.0
95.8
41.7Run12100
8/8
8/8108/8
8/8
1
3/8
2/8
83/3 means detected 3 times out of 3 tests.

Table 11. Sensitivity test results (M. salivarium)
Spike(CFU/mL)

Total

Run
1

Table 8. Sensitivity test results ( M.hyorhinis)
Positive rate(%)

Table 9. Sensitivity test results (M.orale)
RunSpike(CFU/mL)

4

3
8/8
8/8
5/8

Total

Positiverate (%)

24/24
24/24
10/24

100.0
100.0
41.7

5.

Robustness
Regarding the effect of the differences in the detection devices on the performance of Myco Finder, the performance of Myco Finder

was evaluated when LightCycler 480 (Roche) and GVP9600 (Shimadzu Corporation) were used as real-time PCR devices.The results
showed that using different real-time PCR devices does not affect Myco Finder detection performance.

Table 12. Effect of differences in PCR devices on detection performance
Species name
A. laidlawii
M. arginini
M. fermentans
M. hyorhinis

PCR device

Positive count (10 CFU/mL)

LightCycler 480
GVP 9600

3/3
3/3LightCycler 480

3/3

GVP 9600

3/3LightCycler 480

3/3

GVP 9600

3/3LightCycler 480

3/3

GVP 9600

3/3

M.oraleLightCycler 480GVP 9600

3/3M. pneumoniae

LightCycler 480
GVP 9600

3/3
3/3LightCycler 480
GVP 9600

3/3
3/3

M. salivarium

3/3

3/3 means detected 3 times out of 3 tests.

6.

Equivalence Test – Culture Method

In parallel with the detection of 10 CFU/mL of the seven bacterial species to be detected as shown in Table 2, the same sample (10CFU/mL) was used for detection by the culture method. The conditions as an alternative to the culture method (10 CFU/mL) were met, andthe same sample was detected in the culture method.

検体:細胞培養液液体培地(100 mL)
液体培養期間
3日目

7日目

14日目

平板培地(N=2)14日間平板培養
コロニー観察Sample: Cell culture medium
Day 0
Liquid medium (100 mL)
Day 3
Liquid incubation period
Day 7
Plate culture for 14 days
Colony observation
Day 14
Plate medium (N = 2)5

Figure 1. Overview of culture method

1.
2.
3.

Prepare a test sample of 10 CFU/1 mL cell suspension of Mycoplasma Reference Standard for seven mycoplasma species.
Inoculate 0.2 mL of each sample in a plate medium, and incubate at 37 °C for 14 days ().
Inoculate 10 mL of each sample in 100 mL of Hayflick (Millipore) liquid medium, and incubate at 37 C. On days 3, 7, and 14 of incubation,
extract 0.2 mL from each medium and incubate in a plate medium at 37 °C for 14 days ().

4.
Check for the presence/absence of mycoplasma colonies at the time of  to .
Table 13. Comparison of culture results with NATSpecies name
Day 0

No. of days of liquid culturedays of liquid culture
Day 3

Day 7Day 14

NAT
Judgment(using Myco Finder)

A. laidlawii

Yes

Yes

Yes

Yes++

M. arginini

Yes

YesNoNo++M. fermentans

Yes

Yes

Yes

Yes++M. hyorhinis

Yes

Yes

Yes

Yes++M. orale

YesYesNoNo++M. pneumoniaeNoNoNo
Yes

Yes
Yes

Yes
Yes++++M. salivarium
Positive +:The presence of one or more colonies in the plate medium during any incubation period was considered positive.
M. arginini, M. orale returned negative results after day 7, presumably as a result of a long liquid incubation period and the death of all bacteria that grew.

End6

September 29, 2021

Myco Finder Validation Data
Additional Data

Shimadzu Diagnostics Corporation
Research Department

1.

Overview
To evaluate the performance of the mycoplasma gene detection kit (Myco Finder), validation data was obtained with reference tothe following two information. New data on robustness were obtained between February 2021 and August 2021 and are reported asadditional data.

1. The Japanese Pharmacopoeia,17th Edition (the Ministry of Health, Labour and Welfare Notification No. 64, March 7, 2016)
Reference Information
Mycoplasma negative test for cell substrates used in the manufacture of biotechnology-applied
pharmaceuticals/biologically-derived pharmaceuticals p 2395 -23992. PHARM TECH JAPAN (2016) vol. 32, No. 1, 93 -94

2.

Experimental Conditions
The experimental conditions performed with the real-time PCR devices are shown in Table 1, and the mycoplasma information ofthis validation is shown in Table 2.

Table 1. Myco Finder validation detection criteria
DNA extraction reagent QIAGEN CO., LTD.: QIAamp UCP DNA Micro Kit
Kanto Chemical Co., Inc.: Cica geneusR Total DNA Prep Kit (for tissue)

PCR devices

Bio-Rad Laboratories, Inc.: CFX Connect, CFX Opus

PCR conditions

95 °C, 10 seconds
98℃, 3 seconds}45 cycles
60℃, 1 second
Fluorescence detection wavelength: FAM + ROX (or FAM + HEX)

Judgmentof Analysis performed using the software provided with the PCR device (CFX ManagerTM software):measurement results
Regression method, Baseline Subtracted Curve Fit mode

Table 2. Mycoplasma reference material used for validation
Reference materials purchased from ATCC were used to obtain additional data.

Species nameLot No.

StrainGC (copies/mL) *CFU (CFU/mL)

GC/CFUA. laidlawii

ATCC 23206 - TTR

70023819

5.5 x 109

1.3 x 109

4.2

M. arginini

ATCC 23838 - TTR

70018034

1.0 x1010

1.7 x 1010

0.6

M. fermentans

ATCC 19989 - TTR

70015063

7.5 x 109

7.0 x 109

1.1

M. hyorhinis

ATCC 17981 - TTR

70015024

1.5 x 109

1.9 x 109

0.8

M. orale

ATCC 23714 - TTR

70027393

1.1 x 1010

1.8 x 1010

0.6

M. pneumoniae

ATCC 15531 - TTR

70022921

7.0 x 109

1.0 x 109

7.0

M. salivarium

ATCC 23064 - TTR

70025854

1.2 x 1010

8.6 x 109

1.4

*Copies/mL = [DNA contents (g/mL)] x

106x (0.978 x109)/[genome size]

23.

Additional Robustness Data

3-1. Verification of differences in detection devices
Regarding the effect of the differences in the detection devices on the performance of Myco Finder, the performance of Myco
Finder was evaluated when CFX Connect and CFX Opus were used as real-time PCR devices.The results showed that using CFX
Connect and CFX Opus does not affect Myco Finder detection performance.

Table 3. Effect of differences in PCR devices on detection performance Positive count (10Species name
PCR device
CFU/mL)A. laidlawii
CFX Connect
3/3
M. argininiCFX Connect

3/3

M. fermentans

CFX Connect

3/3

M. hyorhinis

CFX Connect

3/3

M. orale

CFX Connect

3/3

M pneumoniae

CFX Connect

3/3

M. salivarium

CFX Connect

3/3

3/3 means detected 3 times out of 3 tests.

Table 4. Effect of differences in PCR devices on detection performance Species
Positive count (10PCR device
nameCFU/mL)A. laidlawii
CFX Opus
3/3
Margin

CFX Opus

3/3

M fermentans

CFX Opus

3/3

M. hyorhinis

CFX Opus

3/3

M. orale

CFX Opus

3/3

M pneumoniae

CFX Opus

3/3

M. salivarium

CFX Opus

3/3

3/3 means detected 3 times out of 3 tests.

3-2. Verification of DNA extraction reagent
Regarding the effect of the differences in DNA extraction reagents on the performance of Myco Finder, the performance of Myco
Finder was evaluated by using the Cica geneusR Total DNA Prep Kit as a DNA extraction reagent. The results showed that theperformance of the Myco Finder was not affected by the use of the Cica geneusR Total DNA Prep Kit.

3Table 5. Effect of differences in DNA extraction reagents on detection performance
Species nameA. laidlawii
M. arginini
M. fermentans
M. hyorhinis
M. oraleM. pneumoniae
M. salivarium

Worker A

Positive countWorker B

Worker C

3/3
3/3

3/3
3/3

3/3
3/3

3/3

3/3

3/3

3/3

3/3

3/3

3/3

3/3

3/3

3/3

3/3

3/3

3/3

3/3

3/3

3/3 means detected 3 times out of 3 tests.

End4