Prepared in November 2015 (1st edition)

Attachment 3
Mycoplasma testing

Mycoplasma Gene
Detection Kit Preversion
Instruction――― Background to Development and Product Features ―――
This kit offers a real-time PCR-based test to detect mycoplasma genes extracted fromcell cultures. As this kit contains two types of strips in which all the reaction reagents aresolidified, reagent preparation is very easy and result can be obtained quickly with easy
operation.
――― General Precautions ―――
1) Read this instructions carefully before using the kit. Follow the operational
procedures specified in this instructions when using the kit. Reliability of the kit
cannot be guaranteed when it is used without following the specified operational
procedures or for any purposes other than those specified in the instructions.
2) All samples should be treated as infectious, and a user has to wear protections(glasses, gloves, masks, etc.) and operate very carefully when using this kit.
3) This reagent is intended for research use only. Do not use this kit for other purposes.
――― Forms and configurations, etc. <Kit components> ―――
Constituent reagents
1．
2．
3．
4．
5．

Test strip A (white) ‥‥‥‥‥‥‥‥‥‥‥……………..…………… 6
Test strip B (transparent)‥‥‥‥‥‥‥‥‥………………………… 6
Positive control (2 x 10^3 copies /μL) ‥‥‥‥………………….‥ 1
Negative control (DNase Free Water) ‥‥‥……………‥.‥‥‥ 1
Flat cap――― Purpose of Use ―――
Detection of mycoplasma genes extracted from cell culture.
――― Operational Precautions ―――
1) If probes or primers in the reaction reagents are degraded by nuclease, the test
cannot yield accurate detection. Nuclease contamination can occur not only fromtest tools and instruments, but also from sweats and saliva of the user. Please
operate carefully.
2) To prevent contamination of the sample, it is recommended that the work spacefor sample preparation/DNA extraction and the work space for reaction reagent
preparation are physically separated. If it is difficult, please perform UV
irradiation or clean-up of the work space before moving on to the next operation.
3) After taking test strips out of the aluminum bag, please seal the bag well so thatthe remaining test strips will be stored safely. Please avoid light as they containfluorogenic reagent.
4) When using the test strips, be careful not to drop them from a height or subject
them to strong physical shock.
5) Mix the positive control well and spin it down before use.
6) Be sure to use new disposable tips to minimize the risk of contamination between
samples when dispensing reagents.
7) When handling a real-time PCR system, follow the instructions of the system.
8) As this kit offers a rea-time PCR-based test in which amplification and detection
are performed at the same time, there is no need to perform electrophoresis ofPCR products. Do not take PCR products out of the test strips as it can cause
contamination.

―――Directions of Use/Dosage (Operation Method)―――[Necessary tools]
Please make the following tools and equipment ready as necessary.
Mixer, micro pipet, filter tip (sterilized, DNase/RNase Free), centrifugal separator,centrifuge tube, real-time PCR system[Operation method]
1. DNA extraction method
Following is the extraction method using QIAGEN DNeasy Blood and Tissue Kit.
Other commercially available DNA extraction kits can also be used, but it isrecommended that validation of extraction efficiency, etc. be performed in advance.
1) Add 20μL of Proteinase K and 200μL of Buffer AL to a 200μL of cell culture or a200μL of cell suspension prepared by concentrating cell culture by centrifugation,and mix well by a mixer.
2) Incubate at 56℃ for 15 minutes.
3) Add 200μL of 99 % ethanol and mix well by a mixer.
4) Move all into spin column and centrifuge at 6,000 × g (8,000 rpm) for 1 minute.
5) Place the spin column into a new collection tube and add 500μL of Buffer AW1.
Centrifuge at 6,000 × g (8,000 rpm) for 1 minute.
6) Place the spin column into a new collection tube and add 500μL of Buffer AW2.
Centrifuge at 20,000 × g (14,000 rpm) for 3 minutes.
7) Place the spin column into a new collection tube and centrifuge the empty column
at 20,000 × g (14,000 rpm) for 1 minute.
8) Confirm that here is no remaining liquid in the column.
9) Place the spin column into a separately prepared centrifuge tube and add 100μL ofBuffer AE. Incubate at room temperature (15 to 25℃) for 5 minutes.
10) Centrifuge at 6,000 × g (8,000 rpm) for 1 minute and prepare a reagent using theobtained sample.
2. How to prepare a reagent
1) Take the necessary quantity of test strip A and test strip B out of the aluminumbag.
2) Add 25μL of the sample prepared by DNA extraction to test strip A, and pipet
gently for 20 times to dissolve the solidified reagent. Dilute the sample asnecessary before use.
3) Move all the dissolved sample in test strip A into test strip B, and pipet gently for
20 times to dissolve the solidified reagent.
4) Attach flat cap to test strip B, and detect by real-time PCR.
*When detection is performed using a white tube, first add the sample to test strip B,dissolve, and then move it into test strip A.
3. Detection by real-time PCR
Following is the protocol for BioRad CFX96.
1)
Set the fluorescence detection wavelength of the real-time PCR system to FAM +ROX (or FAM + HEX). Each fluorescence is used for detection of the following.
(1)
Detection of mycoplasma: FAM(2)
Detection of positive control: FAM + ROX (or FAM + HEX)
*Before starting real-time PCR, fluorescent filter settings need to be adjusted todetect several fluorescence. When BioRad CFX96 is used, set the Scan Mode to‘All channels’ to read data from all the installed filters.
2) Set the program of the real-time PCR in reference to the following standard.
Please refer to the instructions of your real-time PCR system for how to change
settings.

――― How to Judge the Measurement Result ―――
Judgment is made using the analysis software for the real-time PCR system.
BioRad CFX96 uses regression method and Background subtracted Curve Fit modefor analysis.
――― Precautions Regarding Judgment―――
1)
When FAM + ROX is set as fluorescence detection wavelength for real-time
PCR, fluorescence intensity of FAM will increase for a mycoplasma-positive
sample, while fluorescence intensity of FAM and ROX will increase at almost
the same cycle quantification value (Cq value) for a positive control.
Fluorescence intensity will not increase for a negative control or negative
sample.
2) When FAM + HEX is set as fluorescence detection wavelength for real-time PCR,
please be aware of any potential leakage of fluorescence from FAM into HEX.
――― Performance ―――
1. Sensitivity/accuracy test
When standard plasmids of M. arginine, M. orale,M. salivarium,M. hyorhinis,M.
fermentans, M. pneunoniae, and Acholeplasma laidlawii (10 copies / test) are used as
positive control, and negative control (DNase Free Water) is used as negative control
for the test using this kit, all the positive controls will yield positive result, and all thenegative controls will yield negative result.
―――Precautions for Use and Handling ―――
１．Precautions for handling (hazard control)
All samples should be treated as infectious, and a user has to wear protections(glasses, gloves, masks, etc.) and operate very carefully when using this kit to avoid
risk of infection.

2. Precautions for use
1) Avoid freezing this product and store under the specified storage condition (2 to8 ℃).
2) Do not use any expired reagents.
3) Do not mix reagents of different production numbers.
4) Do not use this kit for other purposes than those specified here.
3. Precautions for Disposal
1) To prevent contamination, any test strips used for PCR should be sealed in doubled
plastic bags with the flat cap on, and disposed as medical waste following thewaste disposal rules. Do not autoclave PCR products as they may scatter.
2) Wen disposing of reagents, equipment, etc. used for the test, process them asmedical waste, industrial waste, or infectious waste following the relevant
regulations such as Waste Management and Public Cleaning Act and Water
Pollution Prevention Act, etc.
―――Storage/Expiration Date―――[Storage method]Store at 2 to 8℃.
[Expiration date]
3 months from the production date*Expiration date is indicated on the outer package and on the container label.
――― Contact ―――[Contact for logistics inquiries]
Supply Chain Management Center, Shimadzu Diagnostics Corporation
1075-2 Hokunanmoro, Yuki City, Ibaraki, 307-0036 Tel: 0296 (20) 9700
[Contact for scientific inquiries]
Customer Support Desk, Nihon Techno Service Co., Ltd.
1-19-1 Chuo, Ushiku City, Ibaraki, 300-1234 Tel: 029-886-6811
E-mail shiyaku@ntsbio.com

Sold by:

Shimadzu Diagnostics Corporation
3-24-6 Ueno, Taito-ku, Tokyo,110-8736 TEL 03 (5846) 5611 (Main)

Manufactured by: Nihon Techno Service Co., Ltd.
1-19-1 Ushiku City, Chuo, Ibaraki, 300-1234
TEL 029 (886) 6811

15L-MM002