USER MANUALFor Research Use Only

Myco-Sniff-Valid™

Mycoplasma PCR Detection Kit
Validated for sensitive, specific andrapid detection of mycoplasmaCat. No. 093050301
Size: 48 tests
Storage: -20 °C
Revision Date: 2022-01

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USER MANUAL: Myco-Sniff-Valid™ Mycoplasma PCR Detection Kit

INTRODUCTION
Mycoplasma, a genus of bacteria which lack a cell wall, are commonly foundin research laboratories as contaminants in cell culture. This contamination
can come from individuals in the lab or cell culture media ingredients.
Mycoplasma contamination is a serious concern as it affects various cellularbehaviors including metabolism, growth, viability, and morphology, and thus
compromises the validity of experimental results and study data. Up to30~85% of cell cultures may be contaminated by mycoplasma with the main
species comprised of M. orale, A. laidlawii, M. arginini and M. hyorhinis.
Therefore, testing for mycoplasma is an essential quality control step toassure accurate and reproducible results. Traditionally, a direct culture based
method is used to detect for the presence of contaminating mycoplasma.
However, this method is time-consuming (~28 days), inconsistent, anddifficult to interpret. Recently, PCR-based detection methods have become
an accepted standard protocol for mycoplasma detection to replace direct
culture methods, including European Pharmacopoeia 2.6.7 and USP 63.
Myco-Sniff-Valid™ Mycoplasma PCR Detection Kit has been demonstrated
to be a highly sensitive, specific and rapid method for the detection of
mycoplasma contamination in cell cultures. Myco-Sniff-Valid™ Mycoplasma
PCR Detection Kit is composed of a set of primers, which are specific for thehighly conserved mycoplasma 16S-rRNA coding region, including M.
pneumoniae, M. arginini, M. hyorhinis, M. fermentans, M. orale and A. laidlawii.
This kit is specifically designed to detect the presence of mycoplasma thatmight contaminate biological materials such as cultured cells. The detection
can be performed within 3 hours with a sensitivity as low as 10 CFU/mL.
An exogenous internal control is provided with the Myco-Sniff-Valid
Mycoplasma PCR Detection Kit to distinguish negative reactions resulting
from the absence of mycoplasma contamination from PCR inhibition. The
primer sets included in the kit are used to amplify the internal control andtarget DNA, which are differentiated by size. Furthermore, a positive control
sample is provided with this kit to verify the effectiveness of template DNA
and confirm the size of PCR products for positive samples.
8-Methoxypsoralen (8-MOP) is also included in this kit to prevent crosscontamination by PCR products from previous experiments.
Each kit contains 48 PCR lyophilized tubes for 20 µL reactions.
2KEY BENEFITS
 Highly sensitive: Detection limit as low as 10 CFU/mL.
 Reliable: Meets European Pharmacopoeia Guidance and Regulation.

This test is suitable for product release testing and in-process control.
It can replace culture and indicator cell tests.

 Fast: Replaces traditional 28-day culture testing with a 3 hour PCR test.
 Wide detection range: Detects common cell culture-infecting speciesof mycoplasma and also other various species of mycoplasma (seeTechnical Guide).

 Premixed for ease-of-use: All PCR reaction components are included;
just add template DNA or samples.

 Highly specific: No interference of animal or bacterial DNA.
 Elimination of cross-contamination: 8-MOP prevents crosscontamination from previous PCR products.

 Exogenous internal control: Helps differentiate false negatives dueto PCR inhibition or erroneous PCR tests.

 Sample control: Easily verify the effectiveness of template gDNA bychecking the amplification of the sample control DNA.

3

USER MANUAL: Myco-Sniff-Valid™ Mycoplasma PCR Detection Kit

KIT STORAGE INFORMATIONStore at -20 °C. The kit has a stable shelf life of 1 year without showing anyreduction in performance. The expiration date is labeled on the product boxor Certificate of Analysis.

INTENDED USE
For Research Use Only, not for use in diagnostic procedures.
In-process monitoring for the presence of Mycoplasma. Prior to using it forother purposes, the user must validate the system in compliance withapplicable laws, directives, and regulations.

4KIT CONTENTS/DESCRIPTION
NoContents

Composition

Qty

1

Myco-SniffValid™
Mycoplasma
PCR Premix

0.01% Hot Start Taq DNA Polymerase
0.01% dATP, dTTP, dGTP, dCTP
0.005% Mycoplasma Primers,
Internal Control
0.001% 8-MOP (dissolved in DMSO)

48T

2Control DNA

0.01% genomic DNA extracted from
cultured human cells contaminatedwith M. hyorhinis.

25 μL x 3T

3DNase/RNase FreeWater (D.W.)

No template control:
DNase/RNase Free Water

1 mL x 1T

 Myco-Sniff-Valid™ Mycoplasma PCR Premix: Blue colored pellets in
PCR Strips (48 tests)

 Control DNA: Colorless and transparent liquid
 DNase/RNase Free Water: Colorless and transparent liquid

ADDITIONAL REQUIRED MATERIALS(NOT INCLUDED) Agarose Disposable gloves Pipettes and pipette tips
(aerosol barrier) Electrophoresis equipment
 Thermal cycler
 Vortex mixer

5

USER MANUAL: Myco-Sniff-Valid™ Mycoplasma PCR Detection Kit

OVERVIEW OF MYCOPLASMADETECTION• Sample: 5 μL template + 15 μL D.W. (up to 20 μL)• Positive control: 1 μL positive control DNA + 19 μL D.W.
• Negative control: 20 μL D.W.

6

SAMPLE PREPARATION
Method I: Cell Boiling Method (the most commonly used)
1Prepare cell suspensions from test cell culture in a 1.5 mL tube.
Count cell numbers using standard counting methods. A minimum of
5x104 cells per test are required.
NOTE Strong mycoplasma infections are detected in as little as
10~100 cells, while weak infections require over 5,000~50,000 cells.
Dilute the template according to the suspected infection rates.
We recommend performing the PCR reaction after preparing serial
dilutions of the supernatant to obtain optimal results.

2

Transfer counted cells (over 5x104 cells) to a 1.5 mL tube. Spin the tube ina microcentrifuge for 10~15 seconds. Carefully decant the supernatant.

3

Resuspend the cells in 1 mL of sterile PBS or DPBS solution for washing.

4Spin the tube in a microcentrifuge for 10~15 seconds. Carefully decant
the supernatant. We recommend repeating the wash step once more.

5

Resuspend the cell pellets in 100 μL of sterile PBS or DPBS solution.
NOTE For optimal results, use of PBS solution is recommended over Tris
(10 mM, pH 8.5), TE (10 mM Tris, 0.1 mM EDTA), or autoclaved DW.

6Heat the samples at 95 °C for 10 min, then vortex for 5-10 sec.
Centrifuge for 2 min at 13,000 rpm with a tabletop centrifuge atroom temperature.

7

Transfer an aliquot of the heated supernatant to a fresh tube.
This supernatant will be used as the template in the PCR reaction.

7USER MANUAL: Myco-Sniff-Valid™ Mycoplasma PCR Detection Kit

SAMPLE PREPARATION
Method II: Genomic DNA Extraction

 PCR inhibiting substances may accumulate over time in cell
culture medium.

 Medium with more than 10-12% serum has inhibitory effects on

downstream applications such as PCR. Moreover, phenol red, aroutine material in cell culture medium, is likely to cross-react andthus interfere with the PCR signal detection.

 These negative effects can be overcome by using a general genomic
DNA extraction kit for sample preparation or FastDNA™ SPIN Kit
from MP Bio (SKU 116540600).

 Follow the protocol for genomic DNA extraction kit orFastDNA™ SPIN Kit.

Precautions before PCR Testing Leave the kit at 4 °C or room temperature for thawing. Do not leave itat room temperature more than 1 hour.

 Use clean, disposable gloves when performing the assay and make surethat the work area is clean prior to starting the assay setup.

 All procedures must be performed on a clean bench that should becleaned with 70% alcohol or 10% household bleach (Na-hypochlorite)
after use. Samples should be prepared in a separate area from PCR
reaction setup and use dedicated equipment. Samples are considered
to be biological hazards and must be high-pressure sterilized prior todiscarding.

8

PCR TEST PROTOCOLS
1Prepare the appropriate number of Myco-Sniff-Valid Mycoplasma PCR
Premix tubes. An appropriate number of tubes includes your sample, apositive control and a negative control.

2Add 15 μL of DNase/RNase-free water into the PCR Premix tube.

3Add 5 μL of DNA sample to each of the strip tubes.

4For positive and negative confirmation, use 1 μL of positive control or
DNase/RNase Free water as a test sample. Then, adjust the reaction
volume to 20 μL.

5Dissolve the blue pellet by pipetting or vortexing. The pellet is easily
dissolved by allowing the mixture to stand at room temperature for
1-2 minutes after adding water.

6

Use the thermal cycling conditions presented in the table below forprocessing PCR reactions in a thermal cycler.
PCR Condition

Temp

TimeInitial denaturation

94 °C

1 min

Denaturation

94 °C

30 secAnnealing

60 °C

20 secExtension

72 °C

1 min

72 °C

5 min

X 40 Cycles

Final extension

7For analysis by electrophoresis, use 5 μL from each completed PCR
reaction tube.

8

PCR products should be discarded after UV irradiation (10 min at365 nm) to prevent carry-over contamination.

9

USER MANUAL: Myco-Sniff-Valid™ Mycoplasma PCR Detection Kit

TECHNICAL INFORMATIONInterpretation
Myco-Sniff-Valid Mycoplasma PCR Detection Kit detects variousMycoplasma species with high sensitivity and specificity. For the validationof each PCR reaction, an internal control is included in each PCR Premix
tube. The interpretation of experimental results is as follows:
Band location: Mycoplasma: 260~280 bp, Internal control: 170 bp
MM1122334455

Non specificamplification(ignore)Non specificamplification(ignore)Mycoplasma,
Mycoplasma,
264 ~ 277264
bp ~ 277 bp
Internal
Internalcontrol,control,
170 bp 170 bp
100 bp<(ignore)
PrimerPrimer
dimer, ＜dimer,
100 bp (ignore)

Figure 1. Example Data of Myco-Sniff-Valid Mycoplasma PCR Detection KitLane10Mycoplasma

Test Results

1

Postive

Valid

2

Positive

Valid (Significant amount of target DNA)

3

Negative

Valid (Below 10 CFU/mL)

4

Unknown

Invalid, Retest is needed

5

Negative

Valid (low quality of template DNA or template
degradation; ignore the non specific bands orprimer dimer band)Specificity Test
Myco-Sniff-Valid Mycoplasma PCR Detection Kit is designed to specificallydetect only Mycoplasma spp. and provides high specificity due to a lack ofcross reactivity with other similar microorganisms. Figure 2 shows theevaluation data of Myco-Sniff-Valid Mycoplasma PCR Detection Kit,
suggesting that both internal control (app. 180 bp) and target bands (app.
270 bp) were detected. However, in cases of negative control (20 ng ofnon-mycoplasma bacterial genomic DNA, lanes 2~7) and no template control
samples (lane N), only internal control band was detected.
M1234567NMycoplasma
Internal controlFigure 2. Specificity of Myco-Sniff-Valid Mycoplasma PCR Detection Kit
Lane M: DNA Marker; Lane 1: Mycoplasma hyorhinis; Lane 2: Clostridium perfringens;
Lane 3: Streptococcus mutans; Lane 4: Lactobacillus plantarum; Lane 5: Mobiluncus mulieris;
Lane 6: Gardnerella vaginalis; Lane 7: Haemophilus ducreyi; Lane N: No template control.

LaneName

ATCC No.

1Mycoplasma hyorhinis

17981D-5

2

Clostridium perfringens

13124D-5

3

Streptococcus mutans

700610D-5

4

Lactobacillus plantarum

BAA-793D-5

5

Mobiluncus mulieris

35240D-5

6

Gardnerella vaginalis

49145D-5

7

Haemophilus ducreyi

700724D-5

N

DNase/RNase Free Water

11USER MANUAL: Myco-Sniff-Valid™ Mycoplasma PCR Detection Kit

Analytical SensitivityMyco-Sniff-Valid Mycoplasma PCR Detection Kit is a highly sensitive andoptimal solution for the efficient detection of Mycoplasma spp. contaminationin cultures. To identify the analytical sensitivity, the genomic DNA from 6
Mycoplasma spp. cell cultures was purified. The sensitivity according to theDNA copy number was investigated after purifying gDNA from each cultured
Mycoplasma spp.
Mycoplasma hyorhinis

Mycoplasma oraleM

1234567NM

1

Mycoplasma fermentansM

12345612345634567NM

123456N7N7NAcholeplasma laidlawii
7NM

123456Figure 3. Analytical Sensitivity of Myco-Sniff-Valid Mycoplasma PCR Detection Kit
Lane M: 100 bp DNA Marker; Lane 1: 1×106 CFU/mL of gDNA;
Lane 2: 1×105 CFU/mL of gDNA; Lane 3: 1×104 CFU/mL of gDNA;
Lane 4: 1×103 CFU/mL of gDNA; Lane 5: 1×102 CFU/mL of gDNA;
Lane 6: 10 CFU/mL of gDNA; Lane 7: 1 CFU/mL of gDNA;
Lane N: No template control.

127Mycoplasma pneumoniae

Mycoplasma argininiM2Comparison with direct plating method
Myco-Sniff-Valid Mycoplasma PCR Detection Kit shows much higher
sensitivity than conventional culture plate methods. This is based on thedirect comparison of PCR results performed with this kit to conventional
colony counts using 10-fold dilutions of Mycoplasma culture supernatant.
[Direct plating : A. laidlawii]
Dilution rate

Colony No.

Cell conc. (CFU/mL)

10-7

92 ± 2

0.9×10910-8

12 ± 3.5

1.2×109

10-9

1.3 ± 0.5

1.3×109[PCR Detection : A. laidlawii]
M1234567NMycoplasma
Internal controlFigure 4. Comparative test of Myco-Sniff-Valid Mycoplasma PCR Detection Kit
with direct plating method.
Lane M: DNA Marker; Lane 1: 10-3 diluted A. laidlawii; Lane 2: 10-4 diluted A. laidlawii;
Lane 3: 10-5 diluted A. laidlawii; Lane 4: 10-6 diluted A. laidlawii; Lane 5: 10-7 diluted A. laidlawii;
Lane 6: 10-8 diluted of A. laidlawii; Lane 7: 10-9 diluted A. laidlawii; Lane N: No template control.

13

USER MANUAL: Myco-Sniff-Valid™ Mycoplasma PCR Detection Kit

IMPORTANT NOTES The sequence of amplified PCR products differs slightly from species

to species. You can determine approximately the Mycoplasma species
by sequencing analysis with the following primers. Please refer to thephylogenetic table on pg. 11 of our Myco-Sniff Mycoplasma PCR
Detection Kit User Manual. For more detailed species analysis,
perform additional PCR reactions with your designed primers.

 We list only the Forward primer sequences. Please synthesize theprimer, and then analyze by general sequencing methods.

 Sequencing primer sequence: AGGAT TAG ATA CCC TGG TAG TC-3’(22 mer) The PCR primers used in this kit differ from the sequencing primers.
We do not list the PCR primer sequences contained in this kit.

14

TROUBLESHOOTING GUIDENo Target band in positive reaction
Possible Causes

Comments & Suggestions

Check internalcontrol band

If internal control band is present, thenthe issue is not with the PCR reaction.

Check the quality orconcentration of template

If the PCR reaction is inhibited byimpurities included in the DNA
preparation, the use of diluted DNA
template may be helpful.
If the sample control (app. 570 bp length)
and internal control (app. 160 bp length)
are present and the target band is notvisible, this indicates that the sample is
not infected with mycoplasma.

Check PCR machine

The problem may be caused by the PCRmachine. Check the temperature andensure the machine was programmed
correctly.

15

USER MANUAL: Myco-Sniff-Valid™ Mycoplasma PCR Detection Kit

TROUBLESHOOTING GUIDE (CONT.)
No internal control band
Possible Causes

Comments & Suggestions

Verify templateconcentration

Competition can occur by using highly
concentrated DNA template. Please
repeat the PCR with a diluted template.
If the concentration of template is above
50 ng, the signal of the internal control
may be masked due to competition withthe template. However, the signal of thesample control can still function to serveas an internal control.

Check the quality oftemplate (for the possibilityof contamination with PCRinhibitors)

If the PCR reaction is inhibited byimpurities included in the DNA
preparation, the use of diluted DNA as atemplate may be helpful. If the internalcontrol band is absent, please inquire withour technical support staff.

Check the storage
conditions of the product

Maintain appropriate preservation
conditions.

16

Presence of amplified product in the negative control
Possible Causes

Comments & Suggestions

Check contaminationof D.W.

D.W. may have become contaminated.
Repeat PCR with fresh sterile water.

Check contaminationof lab instruments andenvironments

We recommend using filter tips to reduce
contamination and to sterilize pipettes
prior to use. All procedures should be
performed in dedicated environments freeof contaminants.

Poor resolution on agarose gel
Possible Causes

Comments & Suggestions

Low gel concentration

We recommend to use a 1.5~2% agarosegel. Check the resolution comparing withDNA marker.

Short running time

We recommend that electrophoresis isperformed for 40 min at 100 V/14 cmusing a 6 cm long 2% agarose gel.

17USER MANUAL: Myco-Sniff-Valid™ Mycoplasma PCR Detection Kit

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18

NOTES

19

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