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									Lonza BioResearch

Rapid Mycoplasma Testing

June 2012

n   Mycoplasma – An Introduction

n   Mycoplasma Testing for Research

n   Other tools

                                      slide 2
What Contaminations Can Be Seen in
the Microscope?

Bacteria                 Yeast                    Fungus                Mycoplasma
                         Oval/round in shape
Small black specks                                Filamentous strands   Invisible
                         Smaller than cells
pH change                                         Web-like mesh         Only visible via electron
                         Reflect light (“bright                         microscopy
Often mistaken as cell   beads”)
                         Formed branched chains
Definite movement

                                                                                           slide 3
What Are Mycoplasma?

n   Smallest, simplest prokaryotes
     n   Size ranges from 0.2 to 0.8 µm
     n   Many species cannot be removed by
     n   Cannot be visualized even at very
         high concentrations

n   Lack of rigid cell wall
     n   Not affected by traditional antibiotics
         used in cell culture

n   Limited biosynthetic capabilities
     n   Utilize nutrients from “hosts”

                                                   slide 4
Effects of Mycoplasma on Cells

   Increased sensitivity      Inhibition of cell growth    Alteration of DNA
   to inducers of apoptosis                                transfection efficiency

   Chromosomal aberrations                                     Inhibition of cell

   Disruption of nucleic                                       Production of viruses
   acid synthesis                                              compromised

                                                          DNA fragmentation due to
   Changes in cell                                        Mycoplasmal nucleases
   membrane antigenicity          CELL DEATH              NOT apoptosis

                                                                                       slide 5
Mycoplasma Impair Transfection
                                           HeLa cells infected with
   HeLa cells uninfected
                                           Mycoplasma fermantans

                           Program I-013
                              no DNA

                            no program
                              + DNA

                           Program I-013
   57.7%                      + DNA
   GFP+                                                              GFP+

                                              Preliminary data kindly provided by customer

                                                                               slide 6
Mycoplasma – Types & Frequency

                                 slide 7
Mycoplasma – Prevalence & Sources

n   Prevalence
     n   15-35 % of continuous cell lines
     n   5% of early passage cell cultures
     n   1% primary cell cultures

n   Sources
     n   Cross-contamination from infected cultures
     n   Laboratory personnel
     n   Culture reagents (e.g. bovine serum)
     n   Original isolate tissue (<1%)

                                                      slide 8
The Speed of An Infection

                            slide 9

n   Mycoplasma – An Introduction

n   Mycoplasma Testing for Research

n   Other tools

                                      slide 10
Mycoplasma – Classical Detection Methods

Non-luminescence methods
   n Agar culture test (gold standard)
         n   2 – 3 weeks
         n   Often done externally

    n   PCR methods
         n   4-5 hours
         n   Species detection depends on primer set
         n   Also detects dead mycoplasma

    n   Hoechst stain
         n   Time-consuming, poor indicator
         n   Experience required

                                                       slide 11
MycoAlert™ Mycoplasma Detection Kit

n   Unique 20 min bioluminescent assay for mycoplasma detection
    in cell cultures

n   Mechanism: The assay detects the activity of two enzymes found
    in mycoplasma and other mollicutes
     n   Enzymes are associated with energy generation pathways that
         result in ATP synthesis
     n   The enzymes are found in all 6 of the main mycoplasma cell culture
         contaminants and the majority of mollicute species
     n   Being an enzyme assay, the MycoAlert™ Kit only detects viable
     n   The enzymes are not found in eukaryotic cells

                                                                        slide 12
The MycoAlert™ Reaction
          Specific mollicute substrate                         Photinus pyralis
                                                                (The Fire Fly)

                         Mycoplasma enzymes

                        + Luciferin + O2

     + Oxyluciferin + AMP + PPi + CO2

n   Luciferase reaction:
      n   Very sensitive (detection of 50 attomols ATP)
      n   Wide dynamic range (six orders of magnitude)
      n   Amount of light is proportional to the ATP present
      n   Compatible with 96-well and 384-well formats

                                                                         slide 13
MycoAlert™ Protocol

 Substrate for
                 10 min     Read   B
   Lysis,        5 min
 Luciferase,                Read

                          B/A < 0.9       = negative
   100 µl
                          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
                                                                                                     Infected sample*




                     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



                   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:

                                                                 slide 18

n   Cuvette/tube luminometers
     n Single sample throughput
     n E.g. Lucetta™ Luminometer with tailor-made
        MycoAlert™ Mode
n   Plate-reading luminometers
     n Up to 96 samples per plate
     n Can be semi-automated if fitted with reagent
        injectors for high sample throughput
     n But some are not sensitive enough for MycoAlert
n   Scintillation counters can be used in luminescence mode

n   List of MycoAlert® compatible luminometers is available

                                                              slide 19

n   Mycoplasma – An Introduction

n   Mycoplasma Testing for Research

n   Other Tools

                                      slide 20
Summary – Lonza Products

n   Lonza offers a complete product portfolio for managing
    mycoplasma contaminations:

     n   Detection:
          n   MycoAlert™ Kit and MycoAlert™ Plus Kit for basic research
          n   Lucetta™ Luminometer
          n   MycoTOOL® PCR Kit for final industrial release testing
     n   Elimination:
          n   MycoZap™ Elimination Kit
     n   Prevention:
          n   Specific MycoZap™ Antibiotics
          n   MycoZap™ Spray (EU only)

                                                                          slide 21
Support Tools

n   Please contact the Scientific Support Team for additional
    assistance or to request a MycoAlert™ Demo

n   Other helpful tools
     n (for luminometer list)

                                                                slide 22
Thank you for your attention

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