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                VenorGeM®

Mycoplasma Detection Kit

                 

Instruction Manual

1. Reagents and Materials

1.1 Test kit Components

Oligonucleotide primer set and nucleotides Lyophilized primer set and deoxynucleotide triphosphates dATP, dCTP, dGTP and dTTP at optimized concentrations.                                             1 vial, aliquoted for 25 reactions          1 vial red cap

PCR§ 10x reaction buffer, sterile 100 mM Tris-HCl (pH 8.5) 500 mM KCl 20 mM MgCl2 0.01% Gelatin         1 vial blue cap

Positive control DNA Lyophilized DNA-fragment of Mycoplasma orale prepared by PCR§ and therefore non-infectious.         1 vial green cap

Internal control DNA Lyophilized plasmid DNA including mycloplasma-specific primer sequences and an internal sequence of  the HTLV-1 tax gen with a size of approx.191 bp, non infectious.         1 vial yellow cap.

PCR grade Water deionized, DNA-free water for rehydration of the lyophilized nkit components and for the PCR reaction.    1 vial white cap

1.2 Stability and Storage

The kit is stable until the expiry date given in the Guarantee Certificate if stored at +2 to +8°C. Avoid repeated freezing and thawing after rehydrating primers, nucleotides, and controls.

1.3 Supplemental Requirements

PCR§ thermal cycler

mineral oil, if required for the particular thermal cycler used

tubes for the amplification reaction

agarose gel electrophoresis apparatus

microcentrifuge

micropipettes and filtered tips

autoclaved sterile water (we recommend UV treated)

polymerase: The test is designed to work with standard Taq DNA polymerases. A list of polymerases which were tested for good results is given in the appendix of this manual.

2. Introduction

The polymerase chain reaction (PCR§) was established as the method of choice for highest sensitivity in detecting contaminating mycoplasma. VenorGeM® offers a number of outstanding characteristics over other techniques and products used for detection.

High sensitivity: Detection requires as little as 1 to 5 fg of mycoplasma DNA corresponding to 2-5 mycoplasma per sample volume.

Broad detection range, high specificity: The primer set is specific to the highly conserved rRNA operon, or more specifically, the 16S rRNA coding region in the mycoplasma genome. This allows for detection of all Mycoplasma, Acholeplasma, and Ureaplasma species tested so far and usually encountered as contaminants in cell cultures. Eukaryotic and bacterial DNA is not amplified by VenorGeM®.

Simplicity: Only one protocol is needed for the detection of all mycoplasma species.

Fast results: The detection procedure can be performed within 5 hours.

Easy handling of large sample numbers: With VenorGeM® containing optimized solutions and requiring minimal sample preparation, the testing of numerous samples can be easily accomplished with standard lab equipment.

Internal control: VenorGeM® also provides internal control DNA, which can be added to the reaction. When running the PCR with the internal control DNA, a successfully perfomed reaction is indicated by a distinct 191 bp band on the agarose gel.

3. General Information and Application

Cell lines should be pre-cultured in the absence of antibiotics for several days to maximize test sensitivity. Samples should be derived from cultures which are at confluence.

To avoid false positive results, we recommend the use of sterile deionized H2O, if possible UV-irradiated, aerosol-preventive filter tips and gloves.
This kit is used for the detection of Mycoplasma and Acholeplasma contamination in cell cultures and other cell culture derived biologicals. VenorGeM® is intended for research use only. Not for clinical diagnostics or testing of human samples.

4. Protocol Guide

4.1 Preparation of Templates

Templates for PCR analysis are prepared by boiling extracts of cell culture or other biologicals for 5 minutes:

1. Transfer 100 µl of supernatant from the test culture to a sterile microcentrifuge tube. The top should be  sealed tight

to prevent opening during heating. We recommend microcentrifuge tubes with safety-lock tops.

2. Boil or incubate the sample supernatant at 95°C for 5 minutes.

3. Briefly centrifuge (5 seconds) the sample supernatant to pellet cellular debris before adding to the PCR mixture.

4.2 Rehydration of PCR primer set, nucleotides, and positive control DNA

PCR primer set and nucleotides are aliquoted for 25 reactions. Rehydrate with 130 µl sterile deionized H2O, we recommend UV-irradiated H2O.

Rehydrate positive control with 100 µl and internal control with 220µl sterile deionized H2O, we recommend UV-irradiated H2O.

Rehydration procedure: Before rehydrating positive control DNA, internal control DNA and primer sets with nucleotides, briefly centrifuge tubes to ensure that the lyophilized pellet is spun down.

Add the appropriate amount of H2O and allow the tube to sit at room temperature for 5 minutes. Then mix the solution by vortexing a few seconds or by pipetting up and down repeatedly to completely dissolve the DNA. Briefly centrifuge again.

4.3 Amplification without Internal Control DNA:

When setting up reactions, calculations should include positive and negative controls. Total volume per reaction is 50 µl.

Master Mix Solution:

Mix the following components:

5 µl 10x PCR reaction buffer

5 µl primer set and dNTPs

1 U Taq DNA polymerase

sterile deionized H2O to volume, 48 µl

This mixture is for one reaction and should be adjusted for the number of samples to be analyzed.

Preparation of the PCR Reaction Mix:

Aliquot 48 µl of master mix into each PCR reaction tube.

Add 2 µl of boiled supernatant extract (as described above) to PCR reaction tube per sample being tested.

For controls, add 2 ul of DNA template supplied for positive control, and 2 ul of H2O for negative control.

4.4 Amplification with internal Control DNA

When setting up reactions, calculations should include positive and negative controls. Total volume per reaction is 50 µl.

Master Mix Solution:

Mix the following components:

5 µl 10x PCR reaction buffer

5 µl primer set and dNTPs

2 µl of rehydrated internal control DNA

1 U Taq DNA polymerase

sterile deionized H2O to volume, 48 µl

This mixture is for one reaction and should be adjusted for the number of samples to be analyzed.

Preparation of the PCR Reaction Mix:

Aliquot 48 µl of master mix into each PCR reaction tube.

Add 2 µl of boiled supernatant extract (as described above) to PCR reaction tube per sample being tested.

4.5 Thermal Profile:

94°C for 2 min

55°C for 2 min

72°C for 2 min-Cycle 1

94°C for 30 sec

55°C for 1 min

72°C for 1 min-Cycles 2-30

72°C for 4 min final extension, hold at 4°C.

4.6 Test Evaluation

Amplified PCR products are separated and visualized by standard agarose electrophoresis in 1.5 percent gels with 5 µl of each PCR reaction loaded per lane. Gels should be run at normal setting and the bromophenol blue (BPB) band run no further than 2 cm.

A sample containing mycoplasma DNA will produce a distinct 280-bp band as seen with the positive control but absent with uninfected samples. Another band of 80-90 bp in length is sometimes produced by primer self-annealing and can vary in intensity. This band is often present in all lanes, including the negative control, but does not affect the precision or results of the test. If internal control DNA was used, a successfully performed PCR is indicated by a distinct 191 bp band. This band may fade out with increased amount of amplicons formed, caused by mycoplasma DNA loads in the sample of more than 5 x 10 6 copies/ml.

5. Precaution

It is recommended that the reagents of the kit and specimens be handled carefully using established good laboratory practice:

Do not pipette by mouth.

Do not smoke, eat, or drink in aereas in which specimens or kit reagents are handled.

Wear disposable gloves while handling kit reagents or specimens and wash hands thoroughly afterwards.

6. Appendix

Limited Product Warranty

This warranty limits our liability for replacement of this product. No warranties of any kind, express or implied, including, without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided. Minerva Biolabs shall have no liability for any direct, indirect, consequential, or incidential damages arising out of the use, the results of use, or the inability to use this product.

Notice to Purchaser

This product is optimized for use in the Polymerase Chain Reaction („PCR") covered by patents owned by Hoffmann-La Roche, Inc., and F. Hoffmann-La Roche Ltd. („Roche"). No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser of this product. Minerva Biolabs does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license.

§The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffmann-La Roche. Use of the PCR process requires a license.

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