ISO 9001/14001 Certified Company
PCR Detection Series
e-MycoTM Mycoplasma PCR Detection
Kit (for 20μl rxn)
PROTOCOL
Cat. No. INT-25233 (96 tubes)• Recommended Boiling Extract Method
You can use this protocol for just detecting the contamination of mycoplasma.
However, if you want to perform genotyping for detailed determination of
species, please purify the genomic DNA of suspected mycoplasma-infected
cells by using our G-spin™ Genomic DNA Extraction Kit for cell/Tissue (Cat.
No. 17041).
You may use simply this protocol or your other general boiling methods .
DESCRIPTION
Mycoplasma is common and serious contaminants of cell cultures. It has
been shown that 30-87% of cell cultures are infected with mycoplasma. Many
mycoplasma contaminations, particularly in continuous cell lines, grow slowly
and do not destroy host cells but are still able to affect various parameters,
leading to unreliable or false results. These effects include changes in
metabolism, growth, viability, DNA, RNA and protein synthesis, morphology
etc. Testing for mycoplasma is essential quality control tool to assure accurate
research and reliable biotechnological products.
1. Prepare cell suspensions from the test cell culture to a 1.5ml tube.
And then count cell numbers by general counting methods. The test
cells need at least 5x104 cells per one test.
[Note 1] Harvest adherent cells with trypsin-EDTA solution using standard
techniques. Pipet 1 ml of TE-treated adherent cells. Generally, the
suspension cells such as K562, you need not treat TE solution.
Recommend to count the cells. You should prepare at least 5x104
cells per one test (see Technical guide, >50,000 cells are needed
to complete this protocol).
[Note 2] Strong mycoplasma infections are detected in a little as 20~100
cells, while weak infections require cells from over 50,000 cells.
You can dilute the template according to the infection rates
which you suspect. We recommend to perform PCR reaction
after preparing serial dilutions of the straight supernatant to
obtain optimal results.
This e-Myco™ product has primer sets designed to detect the presence of
mycoplasma which might contaminate in biological materials such as cultured
cells. Conventional techniques used to detect mycoplasma involve culturing
samples on selective media, which needs at least a week to obtain results.
Whereas, by performing PCR with this primer sets based on an conserved
16S rRNA, detection results are obtained with a few hours. As the presence
of contaminant mycoplasma can be easily detected by only verifying the
bands of amplified DNA fragments in electrophoresis, there is no need to
prepare probes labeled with radioisotope, etc.
You can determine the species groups of mycoplasma by sequencing
analysis using sequencing primers suggested from this manual. Furthermore,
if you want to know the detailed species, you may perform PCR reaction and
sequencing from your designed primers.
2. Transfer the counted cells (over 5x104 cells) into 1.5ml 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.
4. Spin the tube in a microcentrifuge for 10~15 seconds. Carefully
decant the supernatant.
[Optional] Repeat this wash step one more.
5. Resuspend the cell pellets in 100μl of sterile PBS or DPBS solutions.
[Note] If you want to the best result, using the PBS solution better
thanTris(10mM, pH 8.5),TE(10mM Tris, 0.1mM EDTA), Autoclaved
DW.
6. Heat the samples at 95℃ for 5 min, and vortexing for 5-10 sec. And
then, centrifuge for 2min at 13,000rpm with a table top centrifuge (at
RT).
7. Transfer an aliquot of the heated supernatant to a fresh tube. This
supernatant will be used as the template in the PCR.
8. Add 10μl of the template to e-Myco™ Mycoplasma PCR Detection
tubes , and then resuspend after adding 10μl of sterile water for 20μl
PCR reaction volume.
9. Perform PCR reaction as the following table.
[Note] Recommend to perform one negative control reaction by adding
20μl of sterile water. We recommend to add 1μl of control DNA for
positive control reaction.
Each tube of e-Myco™ Mycoplasma PCR Detection Kit contains all
components for PCR, i-StarTaqTM DNA Polymerase, dNTPs, 1 x buffer and
primers for mycoplasma partial gene amplifications except PCR template. So,
you can add just templates and perform PCR reaction.
STORAGE
Store at -20°C. This e-Myco™ Mycoplasma Detection PCR Kits are novel
dried premix type. We guarantee 6 months at 4oC and 12 months at -20 °C.
CHARACTERISTICS
• Premix type
Æ This e-Myco™ kit contains all components for PCR reaction. You can
add just a template DNA or samples.
• Broad Species Detection
Æ You can detect the five common cell-culture-infecting species of
mycoplasma, and also other various species of mycoplasma (See
Technical Guide).
• Species Determination
Æ You can determine the species of mycoplasma by sequencing from the
amplified PCR products.
CONTENTS
• Control DNA
(genomic DNA from M. fermentans-infected K562)
• e-Myco™ Mycoplasma PCR Detection tube
• PCR Reaction volume
PCR Condition
Initial denaturation
Denaturation
35
Annealing
cycles
Extension
Final extension
2 ㎍ (100ng/μl)
96 tubes
20 μl reaction
e-Myco™ PCR tube (component)
i-StarTaq DNA Polymerase
Chemical stabilizer I
Loading buffer
dNTPs
Tris-HCl (pH 8.3)
KCl
MgCl2
Mycoplasma Primer Sets
2.5 U
1x
1x
250 mM each
10 mM
50 mM
1.5 mM
10 pmol each
Temp.
94oC
94oC
60oC
72oC
72oC
Time
1 min
30 sec
20 sec
1 min
5 min
• PROTOCOL for using genomic DNA as a template
1. Add the purified genomic DNA using the G-spinTM Genomic DNA
Extraction Kit for Cell/Tissue (Cat.No. 17041) as a template to eMyco™ Mycoplasma PCR Detection tubes and then resuspend after
adding sterile water for 20μl PCR reaction volume.
[Note] : Appropriate amounts of DNA template samples
• Genomic DNA : 50ng – 100ng
Dried under iNtRON’s instruction (Patent Pending)
2. Perform PCR reaction as the upper table.
[Note] Recommend to perform one negative control reaction by adding
20μl of sterile water. We recommend to add 1μl of control DNA for
positive control reaction.
APPLICATIONS
The kit is used for the detection mycoplasma species most commonly
encountered in cell cultures including M. arginini, M. fermentans, M. hyorhinis,
M. orale, and Acholeplasma laidlawii. And more, this kit can detect other
various species of mycoplasma (See Technical guide)
iNtRON BIOTECHNOLOGY
-1-
TECHNICAL GUIDE
PRINCIPLE OF MYCOPLASMA DETECTION
SPECIES DETERMINATION BY SEQUENCING ANALYSIS
The newly developed e-Myco™ Mycoplasma PCR Detection Kit is a highly
sensitive PCR assay which detects various Mycoplasma species that may
contaminate cell culture samples. The primer sets primarily allow detection of
major mycoplasma species in cell culture contaminations (M. arginini, M.
faucium, M. fermentans, M. hyorhinis, M. orale) as well as Acholeplasma
laidlawii. Furthermore, you can detect various mycoplasma species by this
kit (see below). It is a quick, simple and reliable and cost-effective method for
regularly monitoring cells for mycoplasma detection.
1) The sequence amplified PCR products differs slightly from species to
species. You can determine approximately mycoplasma species by
sequencing analysis with the following primers. Please refer to the
phylogenetic table on next page. For more detailed species, you should
perform additional PCR reaction with your designed primers.
The primer sets used in the kit are derived from a highly conserved region
within the 16S rRNA gene and can detect very low levels of contamination.
The rRNA gene sequences of prokaryotes, including mycoplasma, are well
conserved, whereas, the lengths and sequences of the spacer region in the
rRNA operon differ from species to species. So, you can determine the
species to species by sequencing analysis.
Æ Sequencing primer sequences :
M. species Forward 5’- GAT TAG ATA CCC TGG TAG TC-3’ (20 mer)
A. laidlawii Forward 5’- GAT ACC CTG G TA GTC CAC GC-3’ (20 mer)
[Note] The PCR primers used in this kit differ from the sequencing primers.
We don’t inform you the PCR primers contained in this kit.
2) We inform you only Forward primer sequences. Please synthesize the
primer, and then analyze by general sequencing method.
PARTIAL SEQUENCES OF MAJOR CONTAMINANTS
IN CELL CULTURE
ANALYTIC INFORMAION
‹ Origin Type
The following sequences are partial sequences of major contaminants in
general cell cultures. You can classify the species by sequencing analysis.
The Table 1 shows the broad species of
Origin
Type
Origin
mycoplasma detected by this kit. As Type
A
Cell
culture
L
Guinea
Pig
shows, this kit can detect broad range of
Human
M
Squirrels
mycoplasma with high specificity and B
Avian
N
Turkey
sensitivity. The name, Mycoplasma, C
Porcine
O
Puma
from the Greek mykes (fungus) and D
Bovine
P
Canine
plasma (formed), was proposed in the E
Ovine
Q
Primates
1950’s. Mycoplasma is a genus of small F
Equine
R
Lion
b a c teria whic h lac k c ell walls . G
Murine
S
Monkey
Many species were purified and H
I
Insect
T
Mink
characterized from various origins such
Goat
U
Hamster
as cell culture, human, bovine, and etc. J
K
Geese
V
Iguana
We classified briefly by the origins.
‹ Target Primers
*
Designed Primer
The target regions of this kit are divided into Type
Standard
seven of M types (M1~M7) and one of A type M1
15CÆT
for detecting the bulk of the species in the M2
16TÆC
genus Mycoplasma. The designed primers are M3
17CÆT
M4
sufficient to detect major contaminants in cell M5
18TÆC
cultures such as M. arginini, M. faucium, M. M6
18TÆC, 20TÆA
fermentans, M. hyorhinis, M. orale, and M7
8TÆC
A.laidlawii as well as other broad species of
A. laidlawii only
A
mycoplasma.
*Not revealed primer sequences
‹ PCR Product Size
The size of DNA fragments amplified the specific
primers in this kit is about 270 bp. However,
practically, the sizes of PCR product differ
slightly from species to species (268bp ~ 277 bp).
You can confirm by sequencing analysis after
T/A vector cloning and other cloning methods.
Type
I
II
III
IV
V
VI
PCR Size
268 bp
269 bp
270 bp
271 bp
272 bp
277 bp
M. arginini
M. faucium
M. fermentans
M. hyorhinis
M. orale
A. laidlawii
1
1
1
1
1
1
GATTAGATAC
GATTAGATAC
GATTAGATAC
GATTAGATAC
GATTAGATAC
GATACCCTGG
CCTGGTAGTC
CCTGGTAGTC
CCTGGTAGTC
CCTGGTAGTC
CCTGGTAGTC
TAGTCCACGC
cacgccgtaa
cacgccgtaa
cacgccctaa
cacgccgtaa
cacgctgtaa
cgtaaacgat
acgatgatca
acgatgatca
acgatgatca
acgatgatca
acgatgatca
gagaactaag
ttagtcggtg
ttagtcggtg
ttagctgatg
ttagttggtg
ttagtcggtg
tgttggccaa
M. arginini
M. faucium
M. fermentans
M. hyorhinis
M. orale
A. laidlawii
61
61
61
61
61
61
gagagttcac
ggagccactg
gggaactcat
gaataatttc
gaaaactact
aaggtcagtg
tgacgcagct
acgcagctaa
cggcgcagct
actaacgcag
gacgcagcta
ctgcagttaa
aacgcattaa
cgcattaaat
aacgcattaa
ctaacgcgtt
acgcattaaa
cgcattaagt
atgatccgcc
gatccgcctg
atgatccgcc
aaatgatccg
tgatccgcct
tctccgcctg
tgagtagtat
agtagtatgc
tgagtagtac
cctgaatagt
gagtagtatg
agtagtacgt
M. arginini
M. faucium
M. fermentans
M. hyorhinis
M. orale
A. laidlawii
101
101
101
101
101
101
gctcgcaaga
tcgcaagagt
gttcgcaaga
atgctcgcaa
ctcgcaagag
acgcaagtat
gtgaaactta
gaaacttaaa
ataaaactta
gagtgaaact
tgaaacttaa
gaaactcaaa
aaggaattga
ggaattgacg
aaggaattga
taaaggaatt
aggaattgac
ggaattgacg
cggggacccg
gggacccgca
cggggatccg
gacgggaacc
ggggacccgc
ggaccccgca
cacaagcggt
caagcggtgg
cacaagcggt
cgcacaagcg
acaagcggtg
caagcggtgg
M. arginini
M. faucium
M. fermentans
M. hyorhinis
M. orale
A. laidlawii
151
151
151
151
151
151
ggagcatgtg
agcatgtggt
ggagcatgtg
gtggagcatg
gagcatgtgg
atcatgttgt
gtttaatttg
ttaatttgaa
gtttaatttg
tggtttaatt
tttaatttga
ttaattcgaa
aagatacgcg
gatacgcgga
aagatacgcg
tgaagatacg
agatacgcgg
gatacacgaa
gagaacctta
gaaccttacc
tagaacctta
cgtagaacct
agaaccttac
aaaccttacc
cccactcttg
cactcttgac
cccactcttg
tacccactct
ccactcttga
aggtcttgac
M. arginini
M. faucium
M. fermentans
M. hyorhinis
M. orale
A. laidlawii
201
201
201
201
201
201
acatccttcg
atcctttgca
acatcttctg
tgacatcttc
catcccctgc
atactctgca
caatgctata
aagctataga
caaagctatg
tgcaaagcta
aaagctatag
aagttcggag
This sequences are
the partial sequence
of PCR products
Table 1. Mycoplasma Species Detected by e-Myco™ Kit
M. species
A. laidlawii
M. adleri
M. agalactiae
M. alkalescenns
M. anseris
M. arginini
M. arthritidis
M. auris
M. bovigenitalium
M. bovirhinis
M. bovis
M. buccale
M. californicum
M. canadense
M. caviae
M. citelli
M. cloacale
Origin
Type
Primer
Type
PCR
Size
A/E
J
F
E
K
A/B
H
F
E
E
E
B
E
E
L
M
N
A
M2
M2
M5
M5
M5
M5
M5
M2
M2
M2
M5
M2
M5
M2
M2
M5
I
III
III
III
II
III
III
III
III
IV
III
II
II
III
III
I
II
M. species
M. columbinasale
M. columbinum
M. equirhinis
M. falconis
M. faucium
M. felifaucium
M. fermentans
M. gallinarum
M. gateae
M. hominis
M. hyorhinis
M. hyosynoviae
M. iguanae
M. indiense
M. iners
M. leopharyngis
M. maculosum
Origin
Type
Primer
Type
PCR
Size
M. species
Origin
Type
Primer
Type
PCR
Size
C
C
G
C
A
O
A
C
P
A
A/D
P
V
Q
C
R
P
M2
M2
M4
M2
M1
M2
M2
M2
M5
M6
M2
M5
M4
M3
M2
M2
M2
II
III
II
IV
I
II
III
III
III
II
V
II
III
II
II
II
II
M. meleagridis
M. moatsii
M. mustelae
M. opalescens
M. orale
M. oxoniensis
M. penetrans
M. primatum
M. pulmonis
M. salivarium
M. spermatophilum
M. sualvi
M. subdolum
M. synoviae
M. verecundum
C
S
T
P
A
U
B
Q
H
A
B
P
G
C
E
M2
M2
M2
M2
M3
M2
M7
M2
M5
M5
M2
M2
M5
M2
M2
II
III
I
III
II
I
VI
III
IV
II
II
III
III
I
I
-2-
TECHNICAL GUIDE
EXPERIMENTAL INFORMATION
TECHNICAL INFORMATION
‹ Sensitivity
1) This e-Myco™ Mycoplasma PCR Detection Kit will provide a sensitive
means to detect mycoplasma contamination in cell lines. Under optimal
conditions, templates derived from supernatants of an infected cell
culture will yield a maximum signal in the PCR, whereas an uninfected
cell line will yield no PCR products. Undoubtedly, there will be variable
in cell numbers, infection amount, and templates that may contribute to
signal differences in your experiments.
2) It is recommended to use the cultured cells cultivated for 3~6 days after
subculturing as a sample for Mycoplasma detection. You may not detect
efficiently mycoplasma infection when you use the cells those are not
cultivated or are shortly cultivated.
3) We only supply a positive control for validating that a polymerasemediated amplification has occurred. We can’t supply an internal primer
for an amplification efficiency, because the origins of mycoplasma are
variable (see Table 1). We recommend to dilute PCR templates or add
more templates in PCR reaction for an optimal detection range (refer to
Fig. 1).
4) The PCR amplification efficiency is varied by Mycoplasma infection
range. Strong Mycoplasma infections are detected in as little as 10~100
cell equivalents, while weak infections require cell equivalents from the
5,000~50,000 range. So, we recommend to plan various cell numbers in
preparing PCR templates from the culture cells by using the boiling
method. Please refer to Fig. 2.
5) If you perform the genetic analysis for determining more detailed species,
please extract the DNA and apply to the PCR process. We recommend
to use our G-spin™ Genomic DNA Extraction Kit for Cell/Tissue (Cat. No.
17041).
PHYLOGENETIC ANALYSIS TABLE
1) Result for the various concentration of template DNA
Fig.1. Mycoplasma detection was performed for genomic DNA
Genomic DNA were isolated from M. fermentans-infected K562 using G-spinTM
Genomic DNA Extraction Kit for Cell/Tissue (17041). The isolated gDNA were serially
diluted for PCR of mycoplasma detection. These results show that it can be applied for
the mycoplasma detection with small quantities such as 6.3pg of gDNA
Lane M, 100bp DNA Marker; lane1, 200ng; lane 2, 100ng; lane 3, 50ng; lane 4, 25ng;
lane 5, 12.5ng; lane 6, 6.3ng; lane 7, 3.2ng; lane 8, 1.6ng; lane 9, 800pg; lane 10,
400pg; lane 11, 200pg; lane 12, 100pg; lane 13, 50pg; lane 14, 25pg; lane 15, 12.5pg;
lane 16, 6.3pg
2) Result for the various cell number
The following Phylogenetic Analysis Table shows the classification based on the
sequence variations of PCR amplified-products. This cluster can be changed by which
sequences are based on. This cluster is just a reference table.
With a suggested sequencing primer, you can approximately determine the species.
For examples, because the cluster between M.fermentans and M. gallinarum is
different, you can simply classify the species with just sequencing analysis.
However, there is no difference in M. agalactiae, M. caviae, M. fermentans, and M.
M. agalactiae opalescens. In this case, you can’t determine the detailed species
with only this kit and a suggested sequencing primer. If
M. caviae
you want to know the detailed species, you
M. fermentans
have to synthesize newly your PCR
M. opalescens
M. gallinarum
primers, and then analyze by
M. adleri
sequencing analysis.
M. bovigenitalium
M. californicum
M. phocirhinis
M. verecundum
M. citelli
Fig.2. Mycoplasma detection was performed using the e-Myco TM kit
method
Mycoplasma detection from cell lysate of M. fermentans-infected K562 using eMycoTM Mycoplasma Detection Kit. The M. fermentans-infected K562 cells were serially
diluted for PCR of mycoplasma detection and then was performed the e-MycoTM Kit ‘s
protocol. These results show that it can be applied for the mycoplasma detection with
small cell numbers such as 12 cells
Lane M, 100bp DNA Marker; lane NC, Negative control; lane1, 2x105; lane 2, 1x105;
lane 3, 5x104; lane 4, 2.5x104; lane 5, 1.25x104; lane 6, 6.25x103; lane 7, 3.125x103;
lane 8, 1.56x103; lane 9, 7.8x102; lane 10, 3.9x102; lane 11, 1.9x102; lane 12, 98; lane
13, 49; lane 14, 24; lane 15, 12
M. oxoniensis
M. synoviae
M. mustelae
M. bovirhinis
M. moatsii
M. sualvi
M. subdolum
M. auris
M. alkalescens
M. anseris
M. hominis
M. cloacale
M. buccale
M. equirhinis
M. canadense
M. arginini
M. gateae
M. hyosynoviae
M. arthritidis
M. faucium
M. salivarium
M. indiense
M. orale
M. falconis
M. iguanae
M. hyorhinis
M. pulmonis
M. penetrans
M. spermatophilum
M. felifaucium
M. maculosum
M. leopharyngis
M. meleagridis
M. iners
M. columbinum
M. columbinasale
M. bovis
M. primatum
M. lipofaciens
-3-
PARTIAL SEQUENCES OF MYCOPLASMA SPECIES AMPLIFIED BY e-Myco™ KIT
> M. meleagridis
> A. laidlawii
> M. columbinasale
GATACCCTGGTAGTCCACGCCGTAAACGATGAGAACTAAGTGTTGGCCAAAAGGT
CAGTGCTGCAGTTAACGCATTAAGTTCTCCGCCTGAGTAGTACGTACGCAAGTAT
GAAACTCAAAGGAATTGACGGGACCCCGCACAAGCGGTGGATCATGTTGTTTAAT
TCGAAGATACACGAAAAACCTTACCAGGTCTTGACATACTCTGCAAAGTTCGGAG
GATACCCTGGTAGTCCACGCGCCGCCCTAAACGATGATCATTAGCTGATGGAAAA
TTCATCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGACGATACGCGTAGAACCTTACCCACTCTTGACATCTTCCGCAACGCTATA
GATTAGATACCCTGGTAGTCCACGCCCTAAACGATGATCATTAGCTGATGGAAAA
TTCGTCGGCGCACGTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGAAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATA
> M. adleri
> M. columbinum
> M. moatsii
GATTAGATACCCTGGTAGTCCACGCCCTAAACGATGATCATTAGCTGATGGGGAA
CTCATCGGCGCAGCTAACGMATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATA
GATACCCTGGTAGTCCACGCCACGCCCTAAACGATGATCATTAGCTGATGGAAAA
TTCATCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATA
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCATTAGTCGCTAGGGAA
CTTAGTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGA
GTGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATA
> M. agalactiae
> M. equirhinis
> M. mustelae
GATTAGATACCCTGGTAGTCCACGCCCTAAACGATGATCATTAGCTGATGGGGAA
CTCATCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATG
GATACCCTGGTAGTCCACGCCACGCCGTAAACGATGATCATTAGTCGGTGGAGAA
TCACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGAG
TGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAA
TTTGAAGATACACGGAAAACCTTACCCACTTTTGACATCCTTTGCAAAACTATAG
GATTAGATACCCTGGTAGTCCACGCTGTAAACGATGATGATTAGCTGATAGGAAC
TATCGGCACAGCTAACGCATTAAATCATCCGCCTGAGTAGTATGCTCGCAAGAGT
GAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTAAT
TTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATAGA
> M. alkalescens
> M. falconis
> M. opalescens
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCATTAGTCGGTGGAGAA
TTCACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGA
GTGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGGAGAACCTTACCCACTCTTGACATCCTTCGCAAAGCTATA
GATACCCTGGTAGTCCACGCCACGCCGTAAACGATGATCATTAGTCGGTGGAAYR
RTTCACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAG
AGTGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTT
AATTTGAAGATACACGGAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTAT
GATTAGATACCCTGGTAGTCCACGCCCTAAACGATGATCATTAGCTGATGGGGAA
CTCATCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATG
> M. anseris
> M. faucium
> M. orale
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCATTAGTCGGTGGAGAA
TCACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGAG
TGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAA
TTTGAAGATACACGGAGAACCTTACCCACTCTTGACATCCTTCGCAAAGCTATAG
GATACCCTGGTAGTCCACGCCACGCCGTAAACGATGATCATTAGTCGGTGGGAGC
CACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGAGT
GAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAAT
TTGAAGATACGCGGAGAACCTTACCCACTCTTGACATCCTTTGCAAAGCTATAGA
GATTAGATACCCTGGTAGTCCACGCTGTAAACGATGATCATTAGTCGGTGGAAAA
CTACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGAG
TGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAA
TTTGAAGATACGCGGAGAACCTTACCCACTCTTGACATCCCCTGCAAAGCTATAG
> M. arginini
> M. felifaucium
> M. oxoniensis
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCATTAGTCGGTGGAGAG
TTCACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGA
GTGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGGAGAACCTTACCCACTCTTGACATCCTTCGCAATGCTATA
GATACCCTGGTAGTCCACGCCACGCCCTAAACGATGATCATTAGCTGATGGAAGA
TTCATCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATG
GATTAGATACCCTGGTAGTCCACGCTGTAAACGATGATGATTAGTTGATGGACAC
CATCGACGCAGCTAACGCATTAAATCATCCGCCTGAGTAGTATGCTCGCAAGAGT
GAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTAAT
TTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATAGA
> M. arthritidis
> M. fermentans
> M. penetrans
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCATTAGTCGGTGGATTA
ATCACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGA
GTGAAACTTAAAGGAATTGACGGGGACCNGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAANNTACGCGGAGAACCTTACCCACTCTTGACATCCTTCGCAAAGCTATA
GATACCCTGGTAGTCCACGCCACGCCCTAAACGATGATCATTAGCTGATGGGGAA
CTCATCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATG
GATTAGATACCCTGGTAGTCCACACCGTAAACGATGGTAATTAAATCTTGGTACG
GGATGTATCAGGATTGCAGTTAACACATTAAATTACCCGCCTGGGTAGTACATTC
GCAAGAATGAAACTCAAACGGAATTGACGGGGACCCGCACAAGTGGTGGAGCATG
TTGCTTAATTCGACGATACACGTAAAACCTTACCTGGGTTTGACATCCTCTGCAA
> M. auris
>M. gallinarum
> M. primatum
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCATTAGTCGGTGGAGAA
TTCACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGA
GTGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGGAGAACCTTACCCACTCTTGACATCCTTCGCAAAGCTATA
GATACCCTGGTAGTCCACGCCACGCCCTAAACGATGATCATTAGCTGATGGGGAA
CTCATCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATA
GATTAGATACCCTGGTAGTCCACGCCCTAAACGATGATCATTAGTTGATGGGGAA
CTCATCGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATG
> M. bovigenitalium
> M. gateae
> M. pulmonis
GATTAGATACCCTGGTAGTCCACGCCCTAAACGTTGATCATTAGCTGATGGGGAA
CTCATCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGACGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATA
GATACCCTGGTAGTCCACGCCACGCCGTAAACGATGATCATTAGTCGGTGGAGAG
TTCACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGA
GTGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGGAGAACCTTACCCACTCTTGACATCCTTCGCAATGCTATA
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCATTAGCTGGTGGAATT
TTTCACTAGCGCAGCTAACGCATTAAATGATCCACCTGAGTAGTATGCTCGCAAG
AGTGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTT
AATTTGACGATACGCGTAGAACCTTACCCACTCTTGACATCCTTCGCAAAGCTAT
> M. bovirhinis
> M. hominis
> M. salivarium
GATTAGATACCCTGGTAGTCCACGCTGTAAACGATGATGATTAGCTGATAGAGAG
GTCTATCGGCGCAGCTAACGCATTAAATCATCCGCCTGAGTAGTATGCTCGCAAG
AGTGAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTT
AATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTAT
GATACCCTGGTAGTCCACGCCACGCCGTAAACGATGATCATTAGTCGGTGGAGAA
TCACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGAG
TGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAA
TTTGAAGATACACGGAAAACCTTACCCACTCTTGACATCCTTCGCAAAGCTATAG
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCATTAGTCGGCAGAGAA
CTGTTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGAG
TAAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAA
TTTGAAGATACGCGGAGAACCTTACCCACTCTTGACATCCTTCGCAAAGCTATAG
> M. bovis
> M. hyorhinis
> M. spermatophilum
GATTAGATACCCTGGTAGTCCACGCCCTAAACGATGATCATTAGTTGATGGGGAA
CTCATCGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATA
GATACCCTGGTAGTCCACGCCACGCCGTAAACGATGATCATTAGTTGGTGGAATA
ATTTCACTAACGCAGCTAACGCGTTAAATGATCCGCCTGAGTAGTATGCTCGCAA
GAGTGAAACTTAAAGGAATTGACGGGAACCCGCACAAGCGGTGGAGCATGTGGTT
TAATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTA
GATTAGATACCCTGGTAGTCCACGCCCTAAACGATGATCATTAGCTGATGGAGAA
TTCATCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAATGCTATG
> M. buccale
> M. hyosynoviae
> M. sualvi
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCATTAGTCGGTGGAGAA
TCACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGAG
TGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAA
TTTGAAGATACACGGAGAACCTTACCCACTCTTGACATCCTTCGCAATACTATAG
GATACCCTGGTAGTCCACGCCACGCCGTAAACGATGATCATTAGTCGGTGGCCAA
TCACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGAG
TAAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAA
TTTGAGGATACGCGGAGAACCTTACCCACTCTTGACATCCTTCGCAAAGCTATAG
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCATTAGTCGCTAGGGAA
CTTAGTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGA
GTGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGATGTTACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATA
> M. californicum
>M. iguanae
> M. subdolum
GATTAGATACCCTGGTAGTCCACGCCCTAAACGTTGATCATTAGCTGATGGGGAA
CTCATCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGCACCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGATNNTACGCGTGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATAG
GATACCCTGGTAGTCCACGCCACGCCGTAAACGATGATCATTAGCCGGTAGGATA
CTTACTGGCGCAGCTAACGCGTTAAATGATCCGCCTGAGTAGTATGCTCGCAAGA
GTGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGAGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCCTGTGCAATGCTATA
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCATTAGTCGGTGGAGCA
TTCACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGA
GTGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGGAGAACCTTACCCACTCTTGACATCCTTCGCAAAGCTATA
> M. canadense
> M. indiense
> M. synoviae
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCATTAGTCGGTGGAGAA
TTCACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGA
GTGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGGAGAACCTTACCCACTCTTGACATCCTTCGCAATGCTATA
GATACCCTGGTAGTCCACGCCACGCTGTAAACGATGATCATTAGTCGGTGGAAAA
CTACTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGAG
TGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAA
TTTGAAGATACGCGGAGAACCTTACCCACTCTTGACATCCCCTGCAAAGCTATAG
GATTAGATACCCTGGTAGTCCACGCTGTAAACGATGATGACTAGTTGATGGAAAC
CATCGACGCAGCTAACGCATTAAGTCATCCGCCTGAGTAGTATGCTCGCAAGAGT
GAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTAAT
TTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATAGA
> M. caviae
> M. iners
> M. verecundum
GATTAGATACCCTGGTAGTCCACGCCCTAAACGATGATCATTAGCTGATGGGGAA
CTCATCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATG
GATACCCTGGTAGTCCACGCCACGCCCTAAACGATGATCATTAGCTGATGGAAAA
TTCATCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATA
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATGATTAGCTGATGGGAAC
CATCGGCGCAGCTAACGCATTAAATCATCCGCCTGAGTAGTATGCTCGCAAGAGT
GAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTAAT
TTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATAGA
> M. citelli
> M. leopharyngis
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATGATTAGCTGATGGACAC
CATCGGCGCAGCTAACGCATTAAATCATCCGCCTGAGTAGTATGCTCGCAAGAGT
GAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTAAT
TTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATAGA
GATACCCTGGTAGTCCACGCCACGCCCTAAACGATGATCATTAGCTGATGGAAAA
EXPERIMENTAL
INFORMATION
TTCGTCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
Distribuito in ITALIA da
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATA
Li StarFish S.r.l.
> M. cloacale
> M. maculosum
GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATCATTAGTCGGTGGAAAA
TCGCTGACGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTATGCTCGCAAGAG
TGAAACTTAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAA
TTTGAAGATACACGGAAAACCTTACCCACTCTTGACATCCTTCGCAAAGCTATAG
GATACCCTGGTAGTCCACGCCACGCCCTAAACGATGATCATTAGCTGATGGAGAA
TTCGTCGGCGCAGCTAACGCATTAAATGATCCGCCTGAGTAGTACGTTCGCAAGA
ATAAAACTTAAAGGAATTGACGGGGATCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTTGAAGATACGCGTAGAACCTTACCCACTCTTGACATCTTCTGCAAAGCTATA
Via Cavour, 35
20063 Cernusco S/N (MI)
telefono 02-92150794
fax 02-92157285
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