My Co Plasma Detection

8/13/2019 My Co Plasma Detection

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Detection of Mycoplasma contamination in

mammalian cell culture


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commonly called mycoplasma or PPLO

(pleuropneumonia like organisms), class of Moll icutes(soft skin)

with the families:

Mycoplasmataceae: Mycoplasma(animal), Ureaplasma(animal)

Acholeplasmataceae: Acholeplasma(animal, plant)

Spiroplasmataceae: Spiroplasma(plant, rodent)

Anaeroplasma(stomachs of ruminants)

Discovered in 1898 and classified as virus


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The majority of mycoplasma species contaminate

human cell lines

M. arginin i( bovine )

M. fermentans ( human )

M. hyo rhinis ( porcine )

M. orale ( human )

A. laidlawaii ( bovine)


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Differences from other prokaryotes

Lack of cell wall: unable to produce cell wall polymers

Smallest self replicating prokaryotes with coccid

form , 0.3 um

Passfreely through cellulose- and polyvinyl filters with a 0.45 m

pore sizeGenome size is 600 kb to 1700 kb (1/5 of the E. co li genome) with

approximately 500 genes

Does not cause culture turbidity or pH change

Most use alternative UGA=trp code

No TCA cycle and use cholesterol for growth

Parasites for humans, animals, plants, insects


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The Effect of Mycoplasma

Interference of cell growth rate

Induction of morphological alteration ( cyto pathology)

Induction of chromosomal aberration chromosomal aberration

chromosomal aberration

Influencing amino acid and nucleic acid metabolism

Membrane alteration

Transformation

Associate to mammalian cell membranes


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The Effect of Mycoplasma

Fast glucose reduction and formation of acids -> pH waste

Arginine depletion -> inhibition of protein biosynthesis,

cell division and growth

Influence of immunological reactions (macrophage activation,

inhibition of antigen presentation, induction of

signal transduction)

Influence of virus proliferation and the infection rate

Decrease of the transfection rates by 5 % through

electroporation

Induction of leopard cells (condensation of the

chromatins


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Mycoplasma contamination through:

Cross-contamination from untested infected cells toother cell lines

Primary cultures from the original tissue(incidence approximately 4 %) !! tissue graph

Air borne microscopic aerosolization during pipettingTransfer of medium and/or cells during routine

handling when more than one cell line is under the

hood at a time

The same bottle of medium is used for more than onecell line.


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New cultures from unknown sources, also partlyfrom cell banks

Virus suspensions, antibody solutions or otheradditions of contaminated cell cultures


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Prevention:

Good aseptic technique in conjunction with routine

testing.

Always try to work "clean-to-dirty" in order of handling

cultures during a work day or week.

Handle confirmed uncontaminated cells first,

unknown or untested cells next


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Mycoplasma Diagnostic MethodsDNA-binding Fluorescence Coloring Materials

Bisbenzimide, DAPI

Biochemical Verification Methods

Adenosinphosphorylase test (6-MPDR-Test)

Enzyme-Immuno Verification

Cultivation Methods

PCR-Technique


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Mycoplasma Detection

5- primers( Myco-5)

cgc ctg agt agt acg twc gc cgc ctg agt agt acg tac gc

cgc ctg agt agt acg aac gc

tgc ctg rtg agt aca ttc gc tgc ctg atg agt aca ttc gc

tgc ctg gtg agt aca ttc gc

cgc ctg agt agt atg ctc gc cgc ctg agt agt atg ctc gccgc ctg ggt agt aca ttc gc cgc ctg ggt agt aca ttc gc

3-primers( Myco-3)

gcg gtg tgt aca ara ccc ga gcg gtg tgt aca aga ccc ga

Gcg gtg tgt aca aac ccc ga gcg gtg tgt aca aaa ccc ga

r =mixture of a and g gcg gtg tgt aca aac ccc gaW= mixture of t and a


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Extration of genomic DNA

1. Cells harvest from 6 mm dishor 25T flask Wash

with PBS once2. Transfer cells into a clean eppendorf

3. Centrifuge, 2000 rpm, 10 min

4. Discard PBS5. Cell lyse with 300ul cell lysis buffer

6. Add 1.5ul RNAaseA sol , mix by invert 25x

7. Incubate 37o

C, cool to room temperature

8. Add 100 ul protein pricipitation sol

9. Vortex, vigorously, 20sec


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11. Take supernatant to a fresh tube

12. Add 300 ul of Isopropanol, mix by invert 50x till thewhite thread appear

13. Centrifuge, 1 min

14. Wash with 1 ml 75% Ethanol15. Centrifuge, 1 min, and air dry

16. DNA dissolve in 50 ul of 1X T.E buffer

17. Take 1 ugfor PCR detection


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Mycoplasma PCR test

primers Myco-R1 6 ul

Myco-L1 3 ul

dNTP ( 5mM) 2.5 ul

10x Tag buffer 1.5 ul

DNA 2 ul

DDW 0 ul

15 ul

950C 7 min

Tag DNA polymerase 0.5 ul + 1 ul 10x buffer + 8.5ul DDW

65oC 2 min

72oC 5 min

mixture1

mixture2

Pause


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95OC 5 sec

650C 8 sec

720C 16 sec+1(each cycle)

32 cycles

3ul PCR product + 1ul 6X loading dye + 2ul TE

0.7% agarose gel electrophoresis

720C 10min


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Mycoplasma DNA preparation

Cells culture in antibiotic free medium for 2 weeks

Collect 1 ml of supernatant

Centrifuge 13000rpm, 5 min

Resuspend pellet in 1 ml PBS

Centrifuge again 13000rpm, 5 min

Resuspend pellet in 100 ul PBS, heat 95oC, 15 min

Add equal volume of phenol/chloroform, vortex

Take upper layer

Add 2.5 vol of 95% Ethanol, 1/10 vol 3M sodium acetate

-200C , over night

Centrifuge 13000rpm, 10 min

Wash pellet with 1 ml 75% ETOH

Vacuum dry DNA and resuspend DNA in 50ul 1x TE


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Mycoplasma PCR test

primers Myco-R1 6 ul

Myco-L1 3 ul

dNTP ( 5mM) 2.5 ul

10x Tag buffer 1.5 ul

DNA 2 ul

DDW 0 ul

15 ul

950C 7 min

Tag DNA polymerase 0.5 ul + 1 ul 10x buffer + 8.5ul DDW

65oC 2 min

72oC 5 min

mixture1

mixture2

Pause


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95OC 5 sec

650C 8 sec

720C 17 sec

32 cycles

5ul PCR product + 1ul 6X loading dye

1% agarose gel electrophoresis

720C 10min

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My Co Plasma Detection