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Can this system be used for cells in culture, primary
cells, virus stocks and other biologicals?
The Venor®GeMSystem is designed for the detection
of mycoplasmas in cell cultures and virus stocks as well as many other
biological samples. Cell samples may be derived from either cultured cells,
tissue extracts, or directly from cryostocks.
What sample material
can be tested with Venor®GeM?
The prescribed mycoplasma detection protocol employs the testing of the
supernatant. However, other materials that can be tested are Fetal Calf
Serum, vaccines, and paraffin-embedded samples following DNA extraction.
What sample size is
required?
The Venor®GeM System requires as little as 2
µl sample volumen for one reaction. The cells should be cultivated
to 90% confluence to guarantee a maximal density of mycoplasma in the
sample volume.
How sensitive is Venor®GeM?
Venor®GeM is the most sensitive PCR Mycoplasma
Detection Kit available on the market. Detection can be accomplished with
as little as 1-5 fg of mycoplasma DNA that corresponds to 2-5 mycoplasma
per sample volume.
What is the detection
range of Venor®GeM ?
Venor®GeM tests at least for 26 different mycoplasma
species. A complete list of these species is available within this Instruction
Manual. For further information pertaining to the detection range of Venor®GeM,
a downloadable version (pdf format) of the Interlaboratory Validation
Study is available on our website.
Is the intensity of
the PCR amplicon proportional to the contamination level?
Venor®GeM for conventional PCR is a qualitative
test for mycoplasma contamination. A band at between 265-278 bp depicts
a positive result regardless of its intensity. A band of weak intensity
represents a low contamination level, whereas a band of greater intensity
represents a high contamination level. For quantification of the mycoplasma
contamination level you should use our Venor®GeM
for RealTime PCR.
The test has resulted
in non-specific bands.
A PCR amplicon at a non-specific band (i.e. 300 bp) represents a positive
result when this non-specific band corresponds in length to the amplicon
of the positive control. If these non-specific bands do not correspond
to the positive control, they then represent a negative result. Venor®GeM
is highly sensitive, and therefore prone to non-specific annealing which
may generate less intensive bands of various lengths. However, such bands
do not indicate positive results. Possible primer self-annealing produces
another band of 80-90 bp in length, but also does not affect the precision
or results of the test.
Can the processed
samples be stored for later analysis?
After heat treating the cell culture supernatant (500 µ l, 10 min,
95°C), samples can be stored for several days at 4°C prior to
analysis. For long-term storage, it is recommended that samples be stored
at -20°C either in their native state or after heat inactivated.
What are the storage
conditions for the PCR products?
The PCR products are stable for one to two days at room temperature.
For longer storage, the PCR products should be kept at -20°C, at which
temperature they can be kept stable for longer than one year.
Is UV irradiated water necessary for the rehydration
of the PCR primer set, nucleotides, and controls?
The water used for rehydration must be free of DNA. We recommend UV-irradiated
water, however freshly distilled water, gamma-irradiated water, or any
other method for obtaining DNA-free water is also acceptable.
What controls should
be performed?
It is highly recommended that both positive and negative control reactions
be performed for each test series. These controls are to insure assay
conditions, as well as biological positive controls. A negative control
reaction, in which the sample volume is replaced by sterile water, should
also be performed.
Is an internal control necessary?
The internal control is necessary to ensure the quality of the PCR. Samples
should be derived from cultures which are at 80-90% confluence. PCR inhibiting
substances may accumulate in the medium of older cultures. The internal
control band at 191 bp ensures a successfully performed reaction with no
inhibition.
Does an internal
control reduce sensitivity?
No, the internal control does not reduce sensitivity. The oligonucleotide
primer set and nucleotides are designed at optimized concentrations.
What type of DNA polymerase can be used?
We highly recommend our reliable hot-start MB Taq DNA Polymerase (Cat.
No. 53-0200) for optimal Venor®GeM performance
and most sensitive results. We cannot guarantee excellent results with
other polymerases.
Can a separate buffer, other than the universal buffer
supplied with the kit, be used?
It is possible to substitute the Venor®GeM PCR
10x reaction buffer with the specific buffer supplied with the Taq
DNA polymerase. However, the magnesium concentration of the buffer
must then be adjusted to 3.0 mM. ann separat über Minerva Biolabs
bezogen werden (Art.-Nr. 53-0200).
Do I need fixative
agents for sample preparation?
Venor®GeM eliminates the use of hazardous chemicals
found in most standard fixatives used in cell culture technique (e.g.,
formaldehyde) that also interfere with the polymerase reaction. Samples
are prepared and stored after a simple heat inactivation of five minutes
at 95°C, which is sufficient for mycoplasma DNA extraction and gives
the best results in testing.
What kind of inhibitory
substances in the media of old cultures interfere with the PCR reaction?
Which inhibitory substances in the media of old cultures exactly interfere
with the PCR reaction is not known. In general, these are metabolites
since fresh sera or media does not interfere with the PCR. Strikingly,
37% of all samples sent to Minerva Biolabs GmbH for testing mycoplasma
contain inhibitory substances. (Note: If the media is yellow, you will
certainly face inhibition of your PCR reaction)
What cell density
in respect to suspension cell lines would be optimal for a PCR test? What
is the general set-up you would recommend?
The supernatant of suspension cell lines (non-adherent) should be applied
to test for mycoplasma. Cells should not be tested, since debris will
interfere with the PCR reaction. With average titers at 10 6 and a maximum
titer at 10 8 you will find sufficient mycoplasma in the supernatant to
guarantee a sensitive PCR.
What differences exist between the sample kit and the
regular kit?
The only differences are the volumes for resuspension and the mastermix-composition.
The sample kit can be used for 5 reactions, whereas the regular kit is commercially
available for 25, 50, 100 and 250 reactions.
Does Mycoplasma penetrans resides intracellularly?
There is no definite answer to this question, since the appearance of
intracellular M. penetrans still needs to be
demonstrated.
The positive control
band is not visible.
The positive control DNA is a "non-sticky" substance and thus may not
remain at the bottom of the tube. Before rehydration of the positive control
DNA (as well as the internal control DNA and the primer nucleotides),
the tubes must be briefly centrifuged before opening. This will ensure
that the lyophilized pellet is brought to the bottom of the tube, and
thus will not be lost when opening.
What is the concentration
of the positive control provided with Venor®GeM
?
The positive control contains amplicon DNA of a small fragment size.
This can not be diluted properly to give reliable results. The positive
control is only for better reading of positive results. The concentration
of the positive control can vary slightly from lot to lot. Additionally,
the material is not in the right conditions to allow a good quantification
according to the high sensitivity of the test system. As our products
are lyophilised for highest stability this condition is not optimal to
recover exact amount of DNA from a tube wall for quantification purposes.
Out of these reasons we offer special quantification standards from different
microorganisms prepared especially for these applications.
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