USER MANUALFor Research Use Only

Myco-SniffTM
Mycoplasma PCR Detection Kit
Sensitive, specific and rapid detection of mycoplasmaCat. No. 093050201
Size: 48 tests
Storage: -20 °C
Revision Date: 2021-03

www.mpbio.com

USER MANUAL: Myco- SniffTM Mycoplasma PCR Detection Kit

INTRODUCTION
Mycoplasma, a genus of bacteria which lack a cell wall, are commonly found inresearch laboratories as contaminants in cell culture. This contamination cancome from individuals in the lab or cell culture media ingredients. Mycoplasma
contamination is a serious concern as it affects various cellular behaviors
including metabolism, growth, viability, and morphology, and thus
compromises the validity of experimental results and study data. Therefore,testing for mycoplasma is an essential quality control step to assure accurate
and reproducible research. Although agar cultures and DNA fluorochrome
staining methods can be used for mycoplasma detection, polymerase chainreaction (PCR) is the established method of choice for high-sensitivity detection.
Myco-Sniff™ Mycoplasma PCR Detection Kit has been demonstrated to be ahighly sensitive, specific and rapid method for the detection of mycoplasma
contamination in cell cultures. This kit greatly simplifies testing and detectionof mycoplasma contamination in cell cultures with results in less than 3 hours.
After PCR, the presence of contaminating mycoplasma can be easily detectedby simply verifying the bands of amplified DNA fragments after gel electrophoresis.
Myco-Sniff Mycoplasma PCR Detection Kit can be used to detect a broader
range of mycoplasma species compared to other commercially available
PCR-based mycoplasma detection kits. The unique and specific primer sets
used in this kit were designed from DNA sequences that code for highly
conserved 16S rRNA from relevant mycoplasma species, thus preventing
amplification of DNA from other sources. The high sensitivity of the kit allows
for the detection of as little as 20 CFU/mL.
An exogenous internal control is provided with the Myco-Sniff Mycoplasma
PCR Detection Kit to distinguish negative reactions resulting from theabsence of mycoplasma contamination from PCR inhibition. The primer sets
included in the kit are used to amplify the internal control and target DNA,
which are differentiated by size. Furthermore, a positive control sample isprovided with this kit to verify the effectiveness of template DNA and confirm
the size of PCR products for positive samples. 8-Methoxypsoralen (8-MOP) isalso included in this kit to prevent cross-contamination by PCR products fromprevious experiments.
Each kit contains 48 PCR lyophilized tubes for 20 µL reactions.
2

KEY BENEFITS
 Highly sensitive: Detection limit as low as 20 CFU/mL.
 Wide detection range of mycoplasma: Detect mycoplasma from over
8 genera, including 209 individual species.

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

 Highly specific: No interference of animal or bacterial DNA.
 Fast: Detection can be achieved within 3 hours.
 Elimination of cross-contamination: 8-MOP prevents
cross-contamination from previous PCR products.

 Exogenous internal control: Helps differentiate false negatives due toPCR inhibition or erroneous PCR tests.

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

KIT STORAGE INFORMATIONStore at -20 °C. The kit has a stable shelf life of a minimum of 1 year without
showing any reduction in performance. The expiration date is labeled on theproduct box or Certificate of Analysis.

INTENDED USE
 For Research Use Only—Not for use in diagnostic procedures.
 In-process monitoring for the presence of Mycoplasma
3

USER MANUAL: Myco- SniffTM Mycoplasma PCR Detection Kit

KIT CONTENTS/DESCRIPTION
No.

Contents

Composition

Quantity1

Myco-Sniff™
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 fromcultured human cells contaminated withM. hyorhinis.

25 μL x 3T

3DNase/RNase FreeWater (D.W.)

No template control : DNase/RNase
Free Water

1 mL x 1T

 Myco-Sniff™ 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 Thermal cycler
 Disposable gloves Vortex mixer Electrophoresis equipment  Pipettes and pipette tips
(aerosol barrier)

4

OVERVIEW OF
MYCOPLASMA DETECTION• Sample:Sample:
5 μL5template
μL template+ 15+μL
15D.W.
μL D.W.
(up (upto 20toμL)
20 μL)• Positive
Positivecontrol:
control:
1 μL1positive
μL positivecontrol
controlDNA
DNA+ 19+μL
19D.W.
μL D.W.
• Negative
Negative
control:
control:
20 μL
20D.W.
μL D.W.

5

USER MANUAL: Myco- SniffTM Mycoplasma PCR Detection Kit

SAMPLE PREPARATION
Method I: Cell Boiling Method (the most commonly used)1

Prepare 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.

62Transfer counted cells (over 5x104 cells) to a 1.5 mL tube. Spin the tube
in a 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.

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 FastDNATM SPIN Kit
from MP Bio (SKU 116540600).

 Follow the protocol for genomic DNA extraction kit orFastDNATM 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 makesure that 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
to discarding.

7

USER MANUAL: Myco- SniffTM Mycoplasma PCR Detection Kit

PCR TEST PROTOCOLS
1

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

2

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

3

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

4

For positive and negative confirmation, use 1 µL of positive control orDNase/ 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.

6Use the thermal cycling conditions presented in the table below forprocessing PCR reactions in a thermal cycler.

PCR Condition

Temp

Time

Initial denaturation

94 °C

1 min

Denaturation

94 °C

30 secAnnealing

58 °C

20 secExtension

72 °C

1 min

72 °C

5 min

X 35 Cycles

Final extension

87

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

8PCR products should be discarded after UV irradiation (10 min at 365 nm)
to prevent carry-over contamination.

TECHNICAL INFORMATIONInterpretation

 Sample control: indicates that the sample preparation and PCR
reactions worked as expected Target band: indicates mycoplasma infection
 Internal control: indicates the absence of PCR inhibition or erroneous
PCR results►►►

Sample control (app 570 bp)
Target (app 260 bp)
Internal control (app 160 bp)

Figure 1. Example Data of Myco-Sniff™ Mycoplasma PCR Detection Kit

Lane

Mycoplasma

Test Case Description

Template
Amount1

Contamination

Optimal1 ~ 50 ng

2FreeOptimal

1 ~ 50 ng

3Free

Excess template> 50 ng4Contamination

Excess template> 50 ng

5Contamination

Small amount of template

1 ng

9

USER MANUAL: Myco- SniffTM Mycoplasma PCR Detection Kit

Minimal amount of genomic DNA detectable►

Sample control►►TargetInternal control

Figure 2. Result of determining minimal required amount of genomic DNA per test.
To determine the minimal required amount of genomic DNA, genomic DNA was isolated from apure culture of M. fermentans-infected K562 cells using genomic DNA extraction kit. The isolated
genomic DNA was serially diluted for PCR detection. The result indicates that the detection limit
with this kit is 10~20 pg of genomic DNA per test.
LaneMN

123456gDNA

100 bp DNA Marker0 ng

100 ng

50 ng

25 ng

12.5 ng

6.3 ng

3.2 ngLane

789101112131415gDNA

1.6 ng800 pg

400 pg200 pg100 pg

50 pg

25 pg

12.5 pg6.25 pg►

Sample control►►

TargetInternal Control

Minimal cell number required½ dilution

Figure 3. Result of determining minimal required cell number per test.
To determine the minimal required cell number, M. fermentans-infected K562 cells were grown inpure culture, serially diluted and tested. The result indicates that the detection limit with this kit is15 cells per test.
Lane

MN

12Cells

100 bp DNA Marker

0

2.5x105

1.2x105345

6.25x104 3.12x104 1.56x104

67.8x103

Lane

78910

1112

13

1415Cells

3.9x103

1.9x103

9x102

4.8x1022.4x102

120

60

30

1510

DETECTABLE MYCOPLASMA STRAINS(8 GENUS/209 SPECIES)
Genus

Species

Acholeplasma (5)Acholeplasma granularum
Acholeplasma laidlawii
Acholeplasma modicum

Acholeplasma morum
Acholeplasma oculi

Anaeroplasma (3)Anaeroplasma abactoclasticum
Anaeroplasma bactoclasticum

Anaeroplasma varium

Asteroleplasma (1)Asteroleplasma anaerobium

Entomoplasma (5)Entomoplasma lucivorax
Entomoplasma luminosum
Entomoplasma melaleucae

Entomoplasma somnilux
Entoplasma ellychniae

Mycoplasma (182)

M. adleri
M. agalactiae
M. agalactiae (strain PG2)
M. agassizii
M. alkalescens
M. alligatoris
M. alvi
M. amphoriforme
M. anatis
M. anseris
M. arginini
M. arthritidis
M. auris
M. bovigenitalium
M. bovirhinis
M. bovis
M. bovoculi
M. buccale
M. columborale
M. conjunctivae
M. corogypsi
M. cottewii
M. cricetuli
M. crocodyli

M. buteonis
M. californicum
M. canadense
M. canimucosale
M. canis
M. capricolum
M. capricolum subsp
capricolum
M. hyopharyngis
M. hyopneumoniae
M. caviae
M. cavipharyngis
M. citelli
M. cloacale
M. coccoides
M. collis
M. columbinasale
M. columbinum
Mycoplasma sp. Ms01
Mycoplasma sp. Ms02
Mycoplasma sp. Ms03
M. hyopneumoniae(strain 232)

11

USER MANUAL: Myco- SniffTM Mycoplasma PCR Detection KitGenusSpeciesMycoplasma (182)

M. cynos
M. dispar
M. edwardii
M. elephantis
M. equigenitalium
M. equirhinis
M. erythrodidelphis
M. falconis
M. fastidiosum
M. faucium
M. felifaucium
M. feliminutum
M. felis
M. fermentans
M. flocculare
M. gallinaceum
M. gallinarum
M. gallisepticum
M. gallopavonis
M. gateae
M. genitalium
M. genitalium G37
M. glycophilum
M. gypis
M. haemocanis
M. haemofelis
M. haemolama
M. haemomuris
M. hominis
M. neurolyticum
M. opalescens
M. orale
M. ovipneumoniae
M. oxoniensis
M. penetransM. phocicerebrale
M. phocidae
M. phocirhinis
M. pirumM. pneumoniae
M. primatum
M. pullorum
M. pulmonis

12Mycoplasma sp. PG50
M. hyopneumoniae(strain 7448)
M. insonsM. hyorhinis
M. lagogenitalium
M. imitans
M. hyosynoviae
M. indiense
M. iguanae
M. iowae
M. iners
M. leocaptivus
M. leonicaptivi
M. leopharyngis
M. lipofaciens
M. lipophilum
M. microti
M. moatsii
M. mobile
M. molare
M. monodon
M. muris
M. mustelae
M. mycoides
M. mycoides subsp. capri
M. mycoides subsp. mycoides LC
M. mycoides subsp. mycoides SC
M. mycoides sunsp. capri
M. zalophi
M. zalophidermidis
Mycoplasma sp. Saa1e
Mycoplasma sp. Z61
Mycoplasma sp. SF12Mycoplasma sp. 07SH-h
Mycoplasma sp. 07SH-p
Mycoplasma sp. 10T3
Mycoplasma sp. 10T4
Mycoplasma sp. 11CL2
Mycoplasma sp. 1220
Mycoplasma sp. 13CL
Mycoplasma sp. 15CL2
Mycoplasma sp. 237IATGenus

Species

Mycoplasma (182)

M. putrefaciens
M. salivarium
M. simbae
M. spermatophilum
M. maculosum
M. meleagridis
M. sphenisci
M. spumans
M. sturniM. sualvi
M. subdolum
M. suisM. synoviae
M. synoviae (strain 53)
M. testudineum
M. testudinis
M. timone
M. verecundum
M. vulturii
M. wenyonii
M. yeatsii
Mycoplasma sp. M209-8
Mycoplasma sp. M221-9
Mycoplasma sp. M222-2
Mycoplasma sp. M222-5
Mycoplasma sp. M26
M. capricolum subsp.
capripneumoni
Mycoplasma sp. China-1
M. capricolum subsp.
capripneumoniae

Mycoplasma sp. 2F1AT
Mycoplasma sp. 34CL
Mycoplasma sp.39CL
Mycoplasma sp. 50587
Mycoplasma sp. 8790CV
Mycoplasma sp. 94630
Mycoplasma sp. A1802T
Mycoplasma sp. ARNO
Mycoplasma sp. ‘bovine group 7‘
Mycoplasma sp. C3T
Mycoplasma sp. China-1
Mycoplasma sp. CSL 4779
Mycoplasma sp.
CSL 7518-lung
Mycoplasma sp. VJC358
Mycoplasma sp. HRC689
Mycoplasma sp. IS2505
Mycoplasma sp. M1
Mycoplasma sp. M200-2
Mycoplasma sp. M209-7
Mycoplasma sp. ovine/ caprineserogroup 11
M. hyopneumoniae (strain J /ATCC 25934)
Mycoplasma. sp. ‘feline
hemotropic Switzerland‘
Mycoplasma sp. Saa1c
Mycoplasma sp. SF9 e

Mesoplasma (3)Mesoplasma entomophilum
Mesoplasma florumMesoplasma lactucae

Spiroplasma (9)Spiroplasma apis
Spiroplasma citri
Spiroplasma CN-5
Spiroplasma DU-1
Spiroplasma DW-1

Spiroplasma gladiatoris
Spiroplasma mirum
Spiroplasma MQ-1
Spiroplasma taiwanense

Ureaplasma (1)Ureaplasma urealyticum

13

USER MANUAL: Myco- SniffTM Mycoplasma PCR Detection Kit

IMPORTANT NOTES The sequence of amplified PCR products differs slightly fromspecies to species. Sequencing analysis can be approximately
determined for mycoplasma species using the primer indicatedbelow. Please refer to the phylogenetic table on the previous page.
For more detailed species analysis, perform additional PCR
reactions with your designed primers.

 Sequencing primer sequences: GGA TTA GAT ACC CTG GTA GTCCAC G-3’ (25 mer). Note: We list only the Forward primer
sequences. Please synthesize the primer, and then analyze bygeneral sequencing methods.

 The PCR primers used in this kit (sequencing is not listed) differ
from the sequencing primer.

 The PCR conditions were optimized to obtain the highest level ofsensitivity of target gene detection. Therefore, the internal controlband or sample control band may sometimes appear faint dependingon the efficiency of target gene amplification. This efficiency isdependent upon the amount of template DNA added to thereaction. Please refer to the following table for additional
information on this dependency.
Lane

Amount of template DNA

Optimal conditions(three bands are visible)

1 ~ 50 ng of template DNA

Masking point of internal control band

above 50 ng of template DNA

Ending point of sample control bandbelow 1 ng of template DNA

Limit of sensitivity in target
gene amplification14

6.3 pg of template DNA

TROUBLESHOOTING GUIDE
Issue

Possible Causes

Comments & Suggestions

Check internalcontrol band

If internal control band ispresent, then the issue is notwith the PCR reaction.

If the PCR reaction is inhibited by
impurities included in the DNA
preparation, the use of diluted
DNA template may be helpful.
No target band inpositive reaction

Check the quality
or concentrationof template

Check PCR machine

If the sample control(app. 570 bp length) and internal
control (app. 160 bp length) arepresent and the target band isnot visible, this indicates thatthe sample is not infectedwith mycoplasma.

The problem may be causedby the PCR machine.
Check the temperature andensure the machine wasprogrammed correctly.

15

USER MANUAL: Myco- SniffTM Mycoplasma PCR Detection Kit

Issue

Possible Causes

Comments & Suggestions

Verify templateconcentration

Competition can occur byusing highly concentrated DNAtemplate. Please repeat thePCR with a diluted template. If
the concentration of template
is above 50 ng, the signal of
the internal control may bemasked due to competition
with the template. However,the signal of the sample control
can still function to serve as aninternal control.

Check the qualityof template (forthe possibility ofcontamination with
PCR inhibitors)

If the PCR reaction is inhibitedby impurities included in theDNA preparation, the use ofdiluted DNA as a template may
be helpful. If the internal control
band is absent, please inquirewith our technical support staff.

Check the storage
conditions of
the product

Maintain appropriate
preservation conditions.

Check contaminationof D.W.

D.W. may have becomecontaminated. Repeat PCR withfresh sterile water.

Check contamination oflab instruments
and environments

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

No internal
control band

Presence of
amplified productin negative control

16

IssuePossible Causes

Check templateconcentration
No sample
control band

Check the sourceof template

Comments & Suggestions

Sample control band may
become undetectable when theconcentration of DNA templateis below 1 ng.
Check the quantity of DNA
template, and adjust the amountof DNA template in 20 µL PCR
reaction to be above 1 ng.

The primer sets included in thiskit can amplify a human-specific
DNA sequence. If the templatesource is not human cells, theamplification of sample control
will not occur.

RELATED PRODUCT INFORMATION
Product NameSize

Cat. No.

Mycoplasma Removal Agent (MRA)

5 mL

093050044

Myco-SniffTM Mycoplasma PCR Detection Kit

48 tests

093050201

Myco-Sniff-ValidTM Mycoplasma PCR Detection Kit

48 tests

093050301

25 Tests

093050401

50 Tests

093050402

Myco-Sniff-RapidTM
Mycoplasma Luciferase Detection Kit

17

USER MANUAL: Myco- SniffTM Mycoplasma PCR Detection Kit

NOTES

18

19

MP BIOMEDICALSAMERICAS: 800.854.0530 | custserv.na@mpbio.com
EUROPE: 00800.7777.9999 | custserv.eur@mpbio.com
APAC: +65 6775.0008 | custserv.ap@mpbio.com

www.mpbio.com