Lonza BioResearch
Rapid Mycoplasma Testing
June 2012
Content

Mycoplasma – An Introduction

Mycoplasma Testing for Research

Other tools
slide 2
What Contaminations Can Be Seen in
the Microscope?
Bacteria
Yeast
Fungus
Mycoplasma
Small black specks
Oval/round in shape
Filamentous strands
Invisible
Web-like mesh
Only visible via electron
microscopy
pH change
Often mistaken as cell
debris
Smaller than cells
Reflect light (“bright
beads”)
Formed branched chains
Definite movement
slide 3
What Are Mycoplasma?

Smallest, simplest prokaryotes




Lack of rigid cell wall


Size ranges from 0.2 to 0.8 µm
Many species cannot be removed by
filtration
Cannot be visualized even at very
high concentrations
Not affected by traditional antibiotics
used in cell culture
Limited biosynthetic capabilities

Utilize nutrients from “hosts”
slide 4
Effects of Mycoplasma on Cells
Increased sensitivity
to inducers of apoptosis
Inhibition of cell growth
Alteration of DNA
transfection efficiency
Chromosomal aberrations
Inhibition of cell
metabolism
Disruption of nucleic
acid synthesis
Production of viruses
compromised
Changes in cell
membrane antigenicity
CELL DEATH
DNA fragmentation due to
Mycoplasmal nucleases
NOT apoptosis
slide 5
Mycoplasma Impair Transfection
Efficiency
HeLa cells infected with
Mycoplasma fermantans
HeLa cells uninfected
Program I-013
no DNA
no program
+ DNA
57.7%
GFP+
Program I-013
+ DNA
23.0%
GFP+
Preliminary data kindly provided by customer
slide 6
Mycoplasma – Types & Frequency
Acholeplasma laidlawii (bovine):
5-20%
Mycoplasma orale (human): 2040%
Mycoplasma hyorhinis (swine):
10-40%
Mycoplasma arginini (bovine):
20-30%
Mycoplasma fermentans
(human): 10-20%
Mycoplasma hominis (human):
10-20%
slide 7
Mycoplasma – Prevalence & Sources

Prevalence




15-35 % of continuous cell lines
5% of early passage cell cultures
1% primary cell cultures
Sources




Cross-contamination from infected cultures
Laboratory personnel
Culture reagents (e.g. bovine serum)
Original isolate tissue (<1%)
slide 8
The Speed of An Infection
MycoAlert™
MycoAlert® Ratio
Ratio
1000
100
10
1
16 h
22 h
40 h
46 h
64 h
70 h
136 h
0.1
Time after innoculation
K562 control
M. hyorhinis
M. salivarium
slide 9
Content

Mycoplasma – An Introduction

Mycoplasma Testing for Research

Other tools
slide 10
Mycoplasma – Classical Detection Methods
Non-luminescence methods
 Agar culture test (gold standard)



PCR methods




2 – 3 weeks
Often done externally
4-5 hours
Species detection depends on primer set
Also detects dead mycoplasma
Hoechst stain


Time-consuming, poor indicator
Experience required
slide 11
MycoAlert™ Mycoplasma Detection Kit

Unique 20 min bioluminescent assay for mycoplasma detection
in cell cultures

Mechanism: The assay detects the activity of two enzymes found
in mycoplasma and other mollicutes




Enzymes are associated with energy generation pathways that
result in ATP synthesis
The enzymes are found in all 6 of the main mycoplasma cell culture
contaminants and the majority of mollicute species
Being an enzyme assay, the MycoAlert™ Kit only detects viable
mollicutes
The enzymes are not found in eukaryotic cells
slide 12
The MycoAlert™ Reaction
Specific mollicute substrate
Photinus pyralis
(The Fire Fly)
Mycoplasma enzymes
ATP
Luciferase
+ Luciferin + O2
LIGHT
+ Oxyluciferin + AMP + PPi + CO2

Luciferase reaction:

Very sensitive (detection of 50 attomols ATP)

Wide dynamic range (six orders of magnitude)

Amount of light is proportional to the ATP present

Compatible with 96-well and 384-well formats
slide 13
MycoAlert™ Protocol
MycoAlert™
Substrate
Specific
Substrate for
Mollicutes
10 min
MycoAlert™
Reagent
Lysis,
Luciferase,
Luciferin
Sample
100 µl
5 min
Read
B
A
Read
B/A < 0.9
= negative
B/A > 1.2
= positive
0.9 < B/A < 1.2 = quarantine
slide 14
MycoAlert™ Kit – Assay Kinetics
MycoAlert™ Reagent added
Reading A
Reading B
+
MycoAlert™ Substrate added
Clean sample
10000
Infected sample*
RLUs
1000
100
10
1
0
2
4
6
8
10
12
14
16
Time (minutes)
*Infected sample = supernatant from K562 cell culture infected for 72 hours with M. hyorhinis
slide 15
MycoAlert™ Kit – Species Testing
Species
Result
Species
Result
Species
Result
Acholeplasma laidlawii
positive
Mycoplasma canadense
positive
Mycoplasma lipophilum
positive
Acholeplasma modicum
positive
Mycoplasma cloacale
positive
Mycoplasma muris
positive
Acholeplasma morum
positive
Mycoplasma conjunctivae
positive
Mycoplasma neurolyticum
positive
Mesoplasma entomophilum
positive
Mycoplasma equirhinis
positive
Mycoplasma opalescens
positive
Mesoplasma florum
positive
Mycoplasma faucium
positive
Mycoplasma orale
positive
Mycoplasma alkalescens
positive
Mycoplasma fermentans
positive
Mycoplasma pirum
positive
Mycoplasma arginini
positive
Mycoplasma gallinacium
positive
Mycoplasma pneumoniae
positive
Mycoplasma arthritidis
positive
Mycoplasma gallisepticum
positive
Mycoplasma primatum
positive
Mycoplasma bovirhinis
positive
Mycoplasma genitalium
positive
Mycoplasma pulmonis
positive
Mycoplasma bovis
positive
Mycoplasma hominis
positive
Mycoplasma salivarium
positive
Mycoplasma bovoculi
positive
Mycoplasma hyopneumoniae
positive
Mycoplasma spermatophilum
positive
Mycoplasma buccale
positive
Mycoplasma hyorhinis
positive
Mycoplasma synoviae
positive
Mycoplasma californicum
positive
Mycoplasma hyosynoviae
positive
Spiroplasma citri
positive
Mollicute species obtained from the National Collection of Type Cultures UK 39 of 118 species in the collection
tested to date
slide 16
MycoAlert™ Results
MycoAlert™ Assay compared to a PCR kit
100
Ratio
10
1
0.1
K562*
U937*
HL60
JURKAT
CHO*
BJAB
COS7*
MycoAlert™
39
32
0.4
0.4
23
0.8
25
PCR
+
+
-
-
+
-
+
* Positive cell lines (infected with M. hyorhinis)
slide 17
MycoAlert™ Kit –
No Interference with Media Components
Influence of different media components on MycoAlert® Results:
ove
's
Isc
EM
EM
py
ruv
ate
So
diu
m
am
ate
Glu
t
ess
en
t ia
la
min
oa
cid
s
ser
um
No
n
20
%
Try
ps
in
/ED
TA
O
DM
S
10
%
Pe
n/S
tre
p
I
1
RP
M
Ratio
10
0,1
Mycoplasma negative
Mycoplasma positive (addition of M. faucium)
slide 18
Instrumentation




Cuvette/tube luminometers
 Single sample throughput
 E.g. Lucetta™ Luminometer with tailor-made
MycoAlert™ Mode
Plate-reading luminometers
 Up to 96 samples per plate
 Can be semi-automated if fitted with reagent
injectors for high sample throughput
 But some are not sensitive enough for MycoAlert®
measurements
Scintillation counters can be used in luminescence mode
List of MycoAlert® compatible luminometers is available
at: www.lonza.com/mycoalert
slide 19
Content

Mycoplasma – An Introduction

Mycoplasma Testing for Research

Other Tools
slide 20
Summary – Lonza Products

Lonza offers a complete product portfolio for managing
mycoplasma contaminations:

Detection:




Elimination:


MycoAlert™ Kit and MycoAlert™ Plus Kit for basic research
Lucetta™ Luminometer
MycoTOOL® PCR Kit for final industrial release testing
MycoZap™ Elimination Kit
Prevention:


Specific MycoZap™ Antibiotics
MycoZap™ Spray (EU only)
slide 21
Support Tools

Please contact the Scientific Support Team for additional assistance
or to request a MycoAlert™ Demo



scientific.support@lonza.com
scientific.support.EU@lonza.com
Other helpful tools


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www.lonza.com/mycoplasma
www.lonza.com/mycoalert (for luminometer list)
www.lonzabio.com/faq
www.lonza.com/webinar-archive
www.lonza.com/mycotool
www.lonza.com/mycotestserv
slide 22
Thank you for your attention