No.

Category 1

Category 2

Question

Answer
PCR systems and analysis
The required items are: real-time PCR system (dualwavelength compatible), DNA extraction kit, micropipette,

1-3

Fundamental item

Tools/devices

Other than the kit, what is required?

filter chip (sterilized DNase/RNase, Free), mixer, heat block,centrifugal separator (20,000 x g compatible), and 1.5 mL
microtube.
Dual wavelength (FAM and HEX or ROX) fluorescentwavelength is required for mycoplasma gene detection of1-4

Fundamental item

Tools/devices

What real-time PCR system fluorescent wavelength is

this kit, which uses the Taqman probe method. Please

required for detection?

confirm that the real-time PCR system you are using canperform fluorescent detection in combination with FAM +HEX or FAM + ROX.

Fundamental item

Detection

Where is the target area?

It is set to bacterial 16S rRNA genes and the spacer area.

Can the Myco Finder only measure the 7 strains
specified by the Japanese Pharmacopoeia?

It is understood that it can measure 142 strains in silico.
The settings are different for each device, so please carefully2-23

Operation

Detection

How should we configure the settings of the real-timeread the operational manual of the system before

PCR system?

configuring it. If you can choose between NORMAL modeand FAST mode, please select FAST mode.
Please set the time to the smallest number that can be set
in the device that you are using. For some devices,

2-24

Operation

Detection

We cannot set the device that I am using to 3 seconds

reference programs are listed on our company's homepage:or 1 second. What should we do?

https://cosmokai.com/img/products/myco/MycoFinderSample2.pdfFor judgments, we recommend automatic judgment using
the analysis software of the device.

2-25

OperationDetection

How are positives and negatives judged? Is there a(If you can set any of the following methods in the analysis

setting for a cut-off value?

screen of the software you are using, please select them:
Regression method, Background subtracted Curve Fit mode,and 2nd Derivative Maximum method.

1©Shimadzu Diagnostics Corporation All rights reserved.

No.

Category 1

Category 2

Question

AnswerNucleic acid extraction
The stability when stored at room temperature is 1 year. For

1-10

Fundamental item

OtherWith regards to the kit from QIAGEN, what are thetemperatures of 25 degrees Celsius or higher,

stability, time period, and price (and running costs)?

cryopreserving the included Proteinase K is preferable. For
the price, please contact QIAGEN or one of their distributors.
In kit validation, tests are conducted using samples of DNA

2-6Operation

Samples

What is the upper limit for the number of cells that canbe used in tests?

extracted from 1 CHO-DG44 cell × 10^6/mL. The amount of
DNA that can be obtained from extraction depends on thetype of cell, so you will need to investigate the optimal
number of cells for the type of cell that you will use.
Mycoplasma infects culture cells, so only a certain
percentage of the contamination is present in the culture
supernate. Therefore, there is the risk of it not being
possible to obtain a sufficient sensitivity when using a

2-7Operation

Samples

Is it possible to perform tests using culture supernate?

culture supernate as a sample. When using a culture
supernate, it is thought that it is necessary to investigate
conditions, of concentration and the like, that will make itpossible to obtain sufficient sensitivity by means ofcentrifugal separation.
In order to remove mycoplasma bacterial cells in cellsuspensions, we recommend performing centrifugal

2-8

Operation

Samples

What are the concentration conditions of samples?

separation at 15,000 rpm (20,000 x g) for 10 minutes. After
concentration, please resuspend using PBS (-) or the likeand perform DNA extraction according to the protocols.

2-9

Operation

SamplesCan skin tissue or the like be used as samples?

Yes, they can, but the method of DNA extraction will bedifferent.
It is possible to perform DNA extraction using general DNA

2-10

Operation

Extraction

Is it possible to use other methods during the DNA
extraction process?

extraction methods of kits from other companies. However,
we recommend that you confirm the sensitivity. Our
company uses the QIAamp UCP DNA Micro Kit from QIAGEN
to perform kit validation.
Please double check the protocols of the extraction kit thatyou are using. Please confirm that there is no precipitation

2-11

Operation

Extractionof the reagent that you used, and that you have added thenecessary amount of reagent, etc. to be used after adding
the ethanol, etc.
We cannot perform extraction correctly. Also, thevolume is remarkably small.

2©Shimadzu Diagnostics Corporation All rights reserved.

No.

Category 1

Category 2

Question

Answer

We cannot perform extraction correctly. Also, the2-12

Operation

volume is remarkably small.

Extraction

It is thought that ingredients contained in the culture
medium can have an effect on the extraction efficiency. Thisproblem can be fixed by replacing the culture medium with
PBS after performing centrifugal separation on the cellsuspension.
The extraction efficiency decreases remarkably if there is an

2-13

Operation

Extraction

excessive number of cells in the sample. Please use theWe could not obtain nucleic acids after adding 10 CFU of
mycoplasma to a solution that does not contain any
2-14

Operation

Extraction

cells, such as a culture medium or physiological saline
solution, and then performing nucleic acid extraction.
What is the cause of this?

appropriate number of cells and perform extraction again.
It is thought that it is possible to extract nucleic acids
contained in samples at or below the extractable amount of
the extraction kit. If you estimate that the amount of nucleic
acids obtained by the QIAamp UCP DNA Micro Kit, which isrecommended by our company, will be 10 ng or less, it isrecommended that you use carrier DNA.

Kit specifications
There are only short strip tubes. If you are using equipment
1-5

Fundamental item

Kit specifications

Are there longer versions of the strip tubes?

that only supports long ones, you use it by dissolving thereagent in the sample using strip A => strip B => long tube,
and then transferring all of it to the long tube.
This kit amplifies and detects DNA sequences which are

1-6

Fundamental item

Kit specifications

Is it possible to determine the strain from the detectioncommon among many types of mycoplasma, etc., so it isresults?

not possible to determine the strain from the detection
results.

1-7

Fundamental item

Kit specifications

Is it possible to quantify the mycoplasma by using theThis kit was developed as a qualitative kit, so the quantitivity

included positive control and making a calibration curve? is not assured.

3©Shimadzu Diagnostics Corporation All rights reserved.

No.

Category 1

Category 2

1-8

Fundamental item

Kit specifications

Question

Answer
As long as there is no contamination of DNase or the like

What is the expiration date after opening a product?

after opening, you can use the product by the expiration
date.

1-9

Fundamental item

Kit specifications

Is it possible to cryopreserve the product?

No, it is not. Please refrigerate the product.
It is possible to use a strip that has been cut. In such an
event, please set an empty PCR tube in an empty space to

2-16

Operation

Measurement

Is it possible to use a strip that has been cut?

maintain balance when supplying to the PCR system. If it
cannot be balanced on the plate, there is the risk of thisaffecting temperature measurement and making it
impossible for PCR reactions to occur correctly.

Japanese Pharmacopoeia validation
1-1

Fundamental item Japanese Pharmacopoeia related

Are there rules regarding the intervals between
validations?

There are no rules concerning the frequency of validations.
For the 7 strains of mycoplasma specified by the JapanesePharmacopoeia, it must be indicated that detection of 10
CFU/mL in A method and 100 CFU/mL in B method is

1-2

Fundamental item Japanese Pharmacopoeia related

What conditions must be fulfilled in order to use asubstitute for A method or B method?

possible through the performance of equivalence tests.
However, this kit has already been fully validated, so, if you
are using it, you can download the basic data from ourhomepage. (For detailed data, please contact a sales
representative.) Also, depending on the user, equipment
validation of the product may be required.
It is necessary to confirm that 10 CFU/mL of the 7 strains of
mycoplasma specified by the Japanese Pharmacopoeia can2-1

Operation

Test methods

What should be done for equipment validations and forbe accurately and reproducibly detected by equipment andvalidations by cell?

by cell. Moreover, it is thought that it is not necessary to
calculate the detection sensitivity by means of statisticalanalysis while using a three-fold serial dilution. Reference 14©Shimadzu Diagnostics Corporation All rights reserved.

No.

Category 1

Category 2

Question

Answer
The Japanese Pharmacopoeia doesn't have any specific rules
concerning the number of tests conducted, but please

2-2

Operation

Test methods

When performing equipment validation, is there aconfirm that the number of tests is sufficient for the purpose

specific number of times that tests must be conducted?

of use of the device and that the performance needed forthe purpose of use can be obtained. For official information,
you can check with PMDA.
The positive control of this kit can be evaluated by extracting
and measuring a sample of the sample (200 uL) before DNA
extraction. The positive control includes positive control

2-3Operation

Test methods

Are there internal controls (internal standards)?

specific sequences other than mycoplasma gene sequences,and it is possible to distinguish with mycoplasma genomic
DNA by detecting two types of fluorescence: FAM and ROX(or FAM and HEX).
Currently, we don't have the 7 standard strains specified as
subject to detection in the Japanese Pharmacopoeia 17thEdition, but it is possible to purchase the 7 strains for which

2-5

Operation

Test methods

Where can standard mycoplasma strains be acquired?

the CFU/mL are known from the American Type Culture
Collection (ATCC) or another source. Also, users culture andmake strains purchased through the National Institute ofTechnology and Evaluation Biological Resource Center(NBRC), etc.

Culture method of mycoplasma strains

This information is noted in the full kit evaluation.

What is the CFU (Colony Forming Unit) measurement
method?
What are the GC/CFU measurement methods?
Is there a permissible range for GC/CFU values?

This information is noted in the full kit evaluation.
This information is noted in the full kit evaluation.
It has been said that the permissible range of QC/CFUvalues is from 1 to 100.

Is it possible to substitute the 7 standard productstrains (viable bacteria) used in equipment validation

Equipment validation by the Japanese Pharmacopoeia has

with, for example, bacteria-derived genomic DNA,

only been confirmed for viable bacteria.

plasmid DNA, or the like?
It can be done using sterile physiological saline solution, but
What is the dilution method of standard products?

we recommend PBS (D-PBS- Code: 045-29795WAKO) or thelike, which have been tested for mycoplasma.

We cannot obtain the required detection sensitivity.
The number of cells of the test reagent is sometimes 1
× 10^6/mL or higher.

Increase N. For example, if N=4, then it will be judged as
positive in the event that 1/4 is positive.
Using up to 2 μg/well of sample DNA, increase the numberof N so as to make detection possible, and use cases where
1/N is positive to judge positives.

5©Shimadzu Diagnostics Corporation All rights reserved.

No.

Category 1

Category 2

Question

Answer
Reagent control
If you select a strip that suits your equipment, you will

2-15

Operation

Measurement

Is it acceptable to reverse the order of samples to beobtain the correct results if you add the samples in the orderadded to a strip?

from A to B or from B to A. Please select the method that issuitable for your PCR system.

2-17

Operation

Detection

What is the maximum amount of DNA in µg per

Investigations at our company have confirmed that there

reaction?

are no hindrances of PCR reactions up to 2µg/reaction.
The concentration of positive control of this kit is 2 x 10^3copies/μL. １μL is added to 24μL of DW or a positive

2-21

Operation

Detection

How much should be put in for positive control during

sample. When diluting before use, please dilute using

PCR reactions?

10ng/ul of MS2 RNA (from Roche), Easy dilution (fromtakarabio), or the like, and do not dilute using a TE buffer orwater.

2-22

Operation

Detection

Is ROX dye for passive reference included in theIt is not included in this product. Also, ROX channels arereagent? Also, can ROX dye for passive reference beused for positive control, so please do not use Rox dye forused?

passive reference.
It is ideal to separate each of the rooms and perform
reagent control and nucleic acid extraction using a clean

What is the degree of cleanliness of the environments
in which inspections are conducted? (Storage andcontrol of reagents, nucleic acid extraction, detection ofamplification products, etc.)

bench. If this is difficult, one way to do it is to perform
reagent control and nucleic acid extraction using separate
clean benches or the like.
However, performing reagent control and nucleic acidextraction on a laboratory table means that the nucleic acids
might spread to the air as aerosol. Therefore, avoiding it will
reduce risks.

Detection(1) Extraction reagent that has mixed in to eluted nucleic
acid solution may hinder PCR reactions. Before performing
2-18

Operation

Detection

elution, thoroughly check that there is no residue of thereagent inside the column. If there is residue, please
remove the residue from the vacuum centrifuge and pipette.
We added the positive control to the cells and evaluated (2) If the amount of DNA put into the PCR is too high, it

2-19

OperationDetection

the extraction and detection steps, but PCR did notoccur. What is the cause of this?

6might hinder PCR reactions. Please decrease the amount ofDNA and perform PCR.

©Shimadzu Diagnostics Corporation All rights reserved.

We added the positive control to the cells and evaluatedthe extraction and detection steps, but PCR did notNo.

Category 1

Category 2

Question

occur. What is the cause of this?

Answer(3) Please recheck your PCR program and florescence
detection settings. Also, if the scale of the Y-axis of the2-20Operationamplification curve in the analysis screen is too large, itDetection

might not be possible to visually confirm the amplification
curve around the area of the lower limit of detection. Pleasechange the scale of the Y-axis as appropriate.
Is it possible to determine if the strain of mycoplasma is The kit detects mycoplasma genomic DNA, so it cannot
viable bacteria or killed bacteria?

determine if the strain is a viable bacteria or killed bacteria.
False positives can result due to contamination. Sources ofcontamination can exist in various places. (1) The extraction
reagent is contaminated. (2) The tip or tube iscontaminated, (3) The culture substrate is contaminated,(4) There is contamination during reagent control. In thecase of (1) and (4), contamination can result due to positive
samples making contact with chips and entering reagents asa result of misoperation. Therefore, in the event that (1) islikely, we recommend performing extraction again using anew reagent. In the case of (2), there are cases wherecontamination occurs during operation and cases whereNegative control changes to positive.

contamination occurs when a PCR tube, strip, or plate after
PCR is put into an autoclave, the autoclave gets
contaminated, and then unused tips and tubes get
contaminated when put into the autoclave. Therefore, we
recommend using tubes, strips, and plates after PCR has
been performed or tips and tubes that do not require anautoclave. In the case of (3), there may be contamination of
mycoplasma DNA in the raw materials of the culture
medium, such as FBS. Therefore, it is necessary check theculture substrate to be used ahead of time, and to selectmaterials in which no contaminants are mixed.
There is a high risk of contamination in opening PCR

We want to check positive PCR products usingelectrophoresis.

products, so you should avoid opening them. Visualconfirmation is not possible in the case of a low
concentration (10 to 100 copies), but it is possible if there
are at least around 10^5 copies.
Reference 1: Pharm tech japan vol32, No.1 (2016) p93

7©Shimadzu Diagnostics Corporation All rights reserved.