rapid mycoplasma testing lonza bioresearch june 2012
TRANSCRIPT
Rapid Mycoplasma Testing
Lonza BioResearch
June 2012
slide 2
Content
Mycoplasma – An Introduction
Mycoplasma Testing for Research
Other tools
slide 3
What Contaminations Can Be Seen in the Microscope?
Bacteria
Small black specks
pH change
Often mistaken as cell debris
Definite movement
Fungus
Filamentous strands
Web-like mesh
Yeast
Oval/round in shape
Smaller than cells
Reflect light (“bright beads”)
Formed branched chains
Mycoplasma
Invisible
Only visible via electron microscopy
slide 4
What Are Mycoplasma?
Smallest, simplest prokaryotes Size ranges from 0.2 to 0.8 µm Many species cannot be removed by
filtration Cannot be visualized even at very
high concentrations
Lack of rigid cell wall Not affected by traditional antibiotics
used in cell culture
Limited biosynthetic capabilities Utilize nutrients from “hosts”
slide 5
Effects of Mycoplasma on Cells
Inhibition of cell growth
Inhibition of cell metabolism
Disruption of nucleic acid synthesis
Chromosomal aberrations
Changes in cell membrane antigenicity
Alteration of DNA transfection efficiency
Increased sensitivityto inducers of apoptosis
DNA fragmentation due to Mycoplasmal nucleases NOT apoptosisCELL DEATH
Production of viruses compromised
slide 6
Mycoplasma Impair Transfection Efficiency
HeLa cells uninfectedHeLa cells infected with Mycoplasma fermantans
Program I-013no DNA
no program+ DNA
Program I-013+ DNA57.7%
GFP+23.0% GFP+
Preliminary data kindly provided by customer
slide 7
Mycoplasma – Types & Frequency
Acholeplasma laidlawii (bovine):5-20%
Mycoplasma orale (human): 20-40%
Mycoplasma hyorhinis (swine):10-40%
Mycoplasma arginini (bovine):20-30%
Mycoplasma fermentans(human): 10-20%
Mycoplasma hominis (human):10-20%
slide 8
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 9
The Speed of An Infection
0.1
1
10
100
1000
16 h 22 h 40 h 46 h 64 h 70 h 136 h
Time after innoculation
Myc
oA
lert®
Ra
tio
K562 control M. hyorhinis M. salivarium
slide 10
Content
Mycoplasma – An Introduction
Mycoplasma Testing for Research
Other tools
slide 11
Mycoplasma – Classical Detection Methods
Non-luminescence methods Agar culture test (gold standard)
2 – 3 weeks Often done externally
PCR methods 4-5 hours Species detection depends on primer set Also detects dead mycoplasma
Hoechst stain Time-consuming, poor indicator Experience required
slide 12
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 13
The MycoAlert™ Reaction
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
Photinus pyralis(The Fire Fly)
Specific mollicute substrate
ATP
LIGHT + Oxyluciferin + AMP + PPi + CO2
Mycoplasma enzymes
Luciferase+ Luciferin + O2
slide 14
B/A < 0.9 = negative
B/A > 1.2 = positive
0.9 < B/A < 1.2 = quarantine
Sample
100 µl
MycoAlert™ Reagent
MycoAlert™ Substrate
Lysis, Luciferase,
Luciferin
Specific Substrate for
Mollicutes
5 min
10 min
Read AReadB
MycoAlert™ Protocol
slide 15
MycoAlert™ Kit – Assay Kinetics
*Infected sample = supernatant from K562 cell culture infected for 72 hours with M. hyorhinis
Time (minutes)
1
10
100
1000
10000
0 2 4 6 8 10 12 14 16
RL
Us
Reading A
+
MycoAlert™ Substrate added
Reading BMycoAlert™ Reagent added
Clean sample
Infected sample*
slide 16
MycoAlert™ Kit – Species Testing
Mollicute species obtained from the National Collection of Type Cultures UK 39 of 118 species in the collection tested to date
Species Result
Mycoplasma lipophilum positive
Mycoplasma muris positive
Mycoplasma neurolyticum positive
Mycoplasma opalescens positive
Mycoplasma orale positive
Mycoplasma pirum positive
Mycoplasma pneumoniae positive
Mycoplasma primatum positive
Mycoplasma pulmonis positive
Mycoplasma salivarium positive
Mycoplasma spermatophilum positive
Mycoplasma synoviae positive
Spiroplasma citri positive
Species Result
Mycoplasma canadense positive
Mycoplasma cloacale positive
Mycoplasma conjunctivae positive
Mycoplasma equirhinis positive
Mycoplasma faucium positive
Mycoplasma fermentans positive
Mycoplasma gallinacium positive
Mycoplasma gallisepticum positive
Mycoplasma genitalium positive
Mycoplasma hominis positive
Mycoplasma hyopneumoniae positive
Mycoplasma hyorhinis positive
Mycoplasma hyosynoviae positive
Species Result
Acholeplasma laidlawii positive
Acholeplasma modicum positive
Acholeplasma morum positive
Mesoplasma entomophilum positive
Mesoplasma florum positive
Mycoplasma alkalescens positive
Mycoplasma arginini positive
Mycoplasma arthritidis positive
Mycoplasma bovirhinis positive
Mycoplasma bovis positive
Mycoplasma bovoculi positive
Mycoplasma buccale positive
Mycoplasma californicum positive
slide 17
0.1
1
10
100
K562* U937* HL60 JURKAT CHO* BJAB COS7*
Rat
io
MycoAlert™ Results
MycoAlert™ Assay compared to a PCR kit
MycoAlert™ 39 32 0.4 0.4 23 0.8 25
PCR + + - - + - +
* Positive cell lines (infected with M. hyorhinis)
slide 18
MycoAlert™ Kit – No Interference with Media Components
0,1
1
10
RP
MI
Pen
/Stre
p
10%
DM
SO
Tryp
sin/
ED
TA
20%
ser
umN
on e
ssen
tial a
min
o ac
ids
Glu
tam
ate
Sod
ium
pyr
uvat
e
EM
EM
Isco
ve's
Ra
tio
Mycoplasma negative Mycoplasma positive (addition of M. faucium)
Influence of different media components on MycoAlert® Results:
slide 19
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 20
Content
Mycoplasma – An Introduction
Mycoplasma Testing for Research
Other Tools
slide 21
Summary – Lonza Products
Lonza offers a complete product portfolio for managing mycoplasma contaminations:
Detection: MycoAlert™ Kit and MycoAlert™ Plus Kit for basic research Lucetta™ Luminometer MycoTOOL® PCR Kit for final industrial release testing
Elimination: MycoZap™ Elimination Kit
Prevention: Specific MycoZap™ Antibiotics MycoZap™ Spray (EU only)
slide 22
Support Tools
Please contact the Scientific Support Team for additional assistance or to request a MycoAlert™ Demo
[email protected] [email protected]
Other helpful tools 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
Thank you for your attention