For sales rep training

Mycoplasma Gene Detection Kit
Myco Finder (Product Code: 69202)
Operation Manual1

Copyright © Shimadzu Diagnostics Corporation All rights reserved.

Kit Composition (Detection Reagent)

For sales rep training

Mycoplasma Gene Detection Kit, Myco Finder(48 tests)[Appearance]

(1)

(2)
[Constituent reagents](1) Test strip A (white)
6(2) Test strip B (transparent)
6(3) Positive control (2 x 10^3copies/μL) 1(4) Negative control (DNase Free Water)
1(5) Flat cap
62A)

C)

B)
(3)

Copyright © Shimadzu Diagnostics Corporation All rights reserved.

(4)

(5)

For sales rep training

Tools and equipment necessary for measurement

 Micro pipet (Several types: so that 5~1000 µL can be measured to be taken)
 Filter tip (Sterilized, DNase･RNase Free) Mixier Heat block (Water Bath can also be used) Centrifuge (Capable of maximum speed 20,000 x g)
 1.5 mL Micro tube
 Real-time PCR system (Capable of detecting FAM + HEX or ROX)
Heat block

Micro pipet

Real-time PCR

Filter tipCentrifuge
14,000 rpm is required

1.5 ｍL Micro tubeMixer
3

Copyright © Shimadzu Diagnostics Corporation All rights reserved.

Reagents for DNA Extraction

For sales rep training

DNA extraction is performed before using the mycoplasma gene detection kit.
● Introduction of QIAGEN QIAamp UCP DNA Micro Kit

Reagents to be used:
A：Proteinase K

E：Buffer AUW2

B：Buffer AUL

F：Buffer AUE

C：QIAamp UCP MinElute Column

G：Collection Tube

D：Buffer AUW1ABC

DEFGNote) Certain reagents need to be prepared before use. Please follow the instructions on the respective bottle.
4Copyright © Shimadzu Diagnostics Corporation All rights reserved.

DNA Extraction Procedure (1)For sales rep training[Extraction using QIAamp UCP DNA Micro Kit (1)]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.
Note) Mix well until the solution is homogenized.
2) Incubate at 56℃ for 15 minutes. After incubation, spin down to get rid of theliquid on the lid3) Add 200μL of 99 % ethanol and mix well by a mixer.
Note) Mix well until the solution is homogenized.

①Proteinase K 20 μL
Buffer AUL 200 μL

99% Ethanol 200 μL③②Mix well
Cell suspension

5Mix well
Incubate at 56℃ for15 minutes

Copyright © Shimadzu Diagnostics Corporation All rights reserved.

DNA Extraction Procedure (2)For sales rep training[Extraction using QIAamp UCP DNA Micro Kit (2)]4) Move all into QIAamp UCP MinElute Column and centrifuge at 6,000 × g (8,000rpm) for 1 minute.

5) Place the QIAamp UCP MinElute Column into a new collection tube and add
500μL of Buffer AW1. Centrifuge at 6,000 × g (8,000 rpm) for 1 minute.

6Copyright © Shimadzu Diagnostics Corporation All rights reserved.

DNA Extraction Procedure (3)For sales rep training[Extraction using QIAamp UCP DNA Micro Kit (3)]6) Place the QIAamp UCP MinElute 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 QIAamp UCP MinElute Column into a new collection tube andcentrifuge the empty QIAamp UCP MinElute Column at 20,000 × g (14,000 rpm)
for 1 minute.

7Copyright © Shimadzu Diagnostics Corporation All rights reserved.

DNA Extraction Procedure (4)For sales rep training[Extraction using QIAamp UCP DNA Micro Kit (4)]8)

Confirm that there is no remaining liquid in the QIAamp UCP MinElute ColumnNote) The liquid in the example below is colored in blue for better visibility but actual liquid is transparent.

9) Place the QIAamp UCP MinElute Column into 1.5mL tube and add 100μL ofBuffer AUE. Incubate at room temperature (15 to 25℃) for 5 minutes. Centrifuge at
6,000 × g (8,000 rpm) for 1 minute and elute the sample.

Flown outSample
8Reagent Preparation (1) (When BioRad PCR is used)

For sales rep training

Preparation of controls○Prearation of positive control⇒Add 1μL of positive control to 24μL of negative control.
○Preparation of negative control⇒Use the 25 μL of negative control as is.

1) Take the necessary quantity of test strip A and test strip B out of the aluminum bag.
2) Peel off the film from test strip B and add 25 μL each of the sample from DNA
extraction, positive control, and negative control into respective wells, and pipet
gently for 20 times to dissolve the solidified reagent. Dilute the sample asnecessary before use.

9Copyright © Shimadzu Diagnostics Corporation All rights reserved.

Reagent Preparation (2) (When BioRad PCR is used)

For sales rep training

3) Peel off the film from test strip A and move all the dissolved sample in test strip B
into test strip A, and pipet gently for 20 times to dissolve the solidified reagent.
Before dissolution

After dissolution4) Attach flat cap to test strip A, and detect by real-time PCR.

* When detection is performed using a transparent tube, first add the sample totest strip A, dissolve, and then move it into test strip B.
10Copyright © Shimadzu Diagnostics Corporation All rights reserved.

Precautions for extraction

For sales rep training

1) When performing the cleanup steps 4) through 7) of the extraction procedure using
QIAamp UCP MinElute Column, be careful not to let the eluted sample in thecollection tube contact the column.

Operate carefully so that the liquid in the collection tube
will not touch the bottom end of the column.

2) When eluting a sample using Buffer AUE, any remaining Buffer AUW2 in thecolumn will interfere with PCR. Check for any remaining Buffer and if there is,remove it by pipetting, etc. without damaging the filter.
Note) The liquid in the picture below is colored in blue for better visibility but actual liquid is transparent.

11Copyright © Shimadzu Diagnostics Corporation All rights reserved.

Disposal of strips after measurement

For sales rep training

The test strips after PCR have to be disposed of with the caps on and sealed in abag, etc. following the rules of the relevant facility.
If PCR product scatters in the work environment, it can cause contamination or falsepositive result.
Make sure to dispose of the test strips used for PCR with their caps on.

12Copyright © Shimadzu Diagnostics Corporation All rights reserved.