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Mycoplasma MDx

e-Myco™ VALiD Mycoplasma PCR Detection Kit

Cat.No Capacity Inquire
25239 48 tests Inquire
PRODUCT INFORMATION
Description

The e-MycoTM VALiD Mycoplasma PCR Detection Kit is product for the verification of mycoplasma contamination in biological materials.

  • • Simple to Use
  •   - This e-Myco™ VALiD Mycoplasma PCR Detection Kit contains all the components for the PCR reaction.
  •   - You just add template DNA or samples.
  • • Speed - 3 hours
  •   - Replace traditional 28 days culture testing with the kit within 3 hours
  • • Smart - internal control and 8-MOP
  •   - Internal control system embedded in the product prevents misjudgment that possibly arises from an erroneous PCR test.
  •   - This kit can eliminate carry-over contamination with 8-MOP activation.
  • • Steady - Broad Species Detection 
  •   - You can detect common cell culture-infecting species of mycoplasma and also other various species of mycoplasma.
  • • Sensitive and reliable
  •   - Highly sensitive PCR test for the detection of Mycoplasmas al least 10 CFU/ml
  •   - Test for validation according to the KFDA testing guidance similar with E.P. 2.6.7 directive.
  •   - This test  is suitable for release testing and in-process control.
  •   - It can replace culture and indicator cell tests.

The maintenance of contamination-free cell lines is essential to cell-based research such as biopharmaceutical production, cell therapy and tissue engineering. Mycoplasma is often not visible and does not respond to antibiotics, and therefore it is a major issue that requires monitoring and early detection. Up to 30~85% of cell cultures may be contaminated with mycoplasmas, the main contaminants being the species M. orale, A. laidlawii, M. arginini and M. hyorhinis. Although these mycoplasmas do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including changes in metabolism growth, viability and morphology, thereby altering the phenotypic properties of the host cells. Traditional detection methods use direct culture method to detect contaminating organisms. But the culture-based methods are time-consuming, requiring as much as 28 days, very laborious and difficult to interpret. Thus recently, it is a trend that PCR-based detection method may be adopted to standard protocol replacing direct culture method including E.P. 2.6.7 directive and drug regulating agencies worldwide. The e-Myco™ VALiD Mycoplasma PCR Detection Kit is composed a set of primers those are specific for the highly conserved mycoplasma 16S-rRNA coding region including M. pneumoniae, M. agninini, M. hyorhinis, M. fermentans, M. orale and A. laidlawii. The kit is design to detect the presence of mycoplasma that might contaminate biological materials such as cultured cells. Also, the kit can be performed in 3 hours, can detect sensitively until 10 CFU/ml and includes internal control for verifying a PCR run as well as positive control DNA. The adopted 8-methoxypsoralen (8-MOP) is used to extinguish the template activity of contaminated DNAs (PCR product). 8-MOP is known to intercalate into double-stranded nucleic acids and form a covalent interstrand crosslink after photo-activation by incident light at wavelength 320-400 nm. Each tube of the e-Myco™ VALiD Mycoplasma PCR Detection Kit provides all-in-one system(FastMix technology), which means all components for PCR is already pre-aliquoted in each PCR tube. All you need to do is just to add template and distilled water for PCR.

 

                                                         [OVERVIEW OF MYCOPLASMA DETECTION]

 

Applications

The kit is used for the detection of mycoplasma species that are most commonly encountered in cell culture, including M. peumoniae, M. arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii. Furthermore, this kit can detect other various species of mycoplasma

Kit Contents
Contents 25239 25240
e-Myco™ VALiD Mycoplasma PCR Premix 48 Test 8 Test
Control DNA
(Recombinant DNA included partial 16S sequence of M. hyorhinis)
20 μl 5 μl
DNase/RNase-free Distilled Water 1 ml 0.2 ml
Technical Data

Analytical Sensitivity

e-Myco™ VALiD Mycoplasma PCR Detection Kit is an eligible kit for the efficient detection of Mycoplasma spp. contamination with high sensitivity in the culture. To identify the analytical sensitivity, the genomic DNA from cell cultured 6 Mycoplasma spp. were purified. Mycoplasma species used for this experiment are as follows.

 

 

Analytical Sensitivity of e-Myco™ VALiD Mycoplasma PCR Detection Kit

The sensitivity according to the DNA copy number was investigated after purifying gDNA from each cultured Mycoplasma spp.

 

 

Fig. Analytical Sensitivity of e-Myco™ VALiD Mycoplasma PCR Detection Kit

LaneM, SiZer™-100 DNA Marker; Lane 1, 1×106 cfu/ml of gDNA; Lane 2, 1×105 cfu/ml of gDNA; Lane 3, 1×104 cfu/ml of gDNA; Lane 4, 1×103 cfu/ml of gDNA; Lane 5, 1×102 cfu/ml of gDNA; Lane 6, 10 cfu/ml of gDNA; Lane 7, 1 cfu/ml of gDNA; Lane N, No template control.

Comparison with direct plating method

e-Myco™ Mycoplasma PCR Detection Kit shows much higher sensitivity than conventional culture plate method, based on the direct comparison of PCR result done by this kit with the conventional colony counts, using 10-folds diluted Mycoplasma culture supernatant

 

Fig. Comparative test of e-Myco™ VALiD Mycoplasma PCR Detection Kit with direct plating method.

LaneM, SiZer™-100 DNA Marker; Lane 1, 10-3  diluted A. laidlawii; Lane 2, 10-4 diluted A. laidlawii; Lane 3, 10-5 diluted A. laidlawii; Lane 4, 10-6 diluted A. laidlawii; Lane 5, 10-7 diluted A. laidlawii; Lane 6, 10-8 diluted of A. laidlawii; Lane 7, 10-9 diluted A. laidlawii; Lane N, No template control

TroubleShooting Guide
QNoTarget band in positive reaction
AIf internal control band is seen, PCR has been performed properly. It is not a problem of the product. If the PCR reaction is inhibited by impurities included in DNA preparation, the use of diluted DNA as a template may be helpful.
QNo internal control band
ACompetition can occur by using high concentrated DNA template. Please repeat the PCR with a diluted template. If the concentration of template is above 50 ng, the signal of internal control may be disappeared by competition with the template.
QPresence of amplified product in the negative control
AD.W. can be contaminated. Perform PCR again with fresh sterile water. Also, we recommend that you use filter tips to reduce contamination and that you use a pipette after sterilization. All procedures should be done in sterilized conditions.
QPoor resolution on agarose gel
AWe recommend to use a 1.5~2% agarose gel. Also, we recommend that electrophoresis is performed for 40 min at 100 V/14 cm using a 6 cm long 2% agarose gel.
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