- Mycoplasma PCR Kit is a ready-to-use system for the detection of Mollicutes (Mycoplasma, Acholeplasma, Spiroplasma, Ureaplasma) DNA in cell cultures, animal tissue samples and other biological matrices by conventional PCR.
- The kit enables the direct detection of all Mollicutes species known as contaminants of cell cultures.
- Mycoplasma PCR Kit contains reagents and primers for the amplification ofthe 16S rRNA coding region in the mycoplasma genome. The mycoplasma-specific amplification is detected at 104-113 bp.
- In addition, the kit contains a second heterologous amplification system to monitor the DNA isolation procedure and identify possible PCR inhibition simultaneously. The internal control system consists of: primers present in Mycoplasma Master Mix (2x) and exogenous DNA fragment (IC-internal control). The amplification of the inner control is detected at 191 bp and does not interfere with the amplification of the specific mycoplasma DNA.
- IC can be added at the DNA isolation stage (point a) – then it is both a control of the DNA isolation and PCR inhibition, or only at the PCR stage (point b) – to assess the quality of the purified DNA and possible PCR inhibition.
- Add IC to the lysis buffer or to the mixture of lysis buffer and sample material (do not add to the sample material directly) at a ratio 0,1 μl per 1 μl elution volume. For example, 5 μl of IC should be added initially using 50 μl elution volume. It is recommended to use carrier RNA when isolating DNA from from cell free body fluids and material low in DNA/RNA content. Carrier RNA increases the efficiency of DNA binding to silica membranes in the presence of small amounts of genetic material in the sample.
- Add 0.5 µL of IC for every 25 µL of PCR reaction.
- The specificity of Mycoplasma PCR Kit has been confirmed by PCR using purified DNA (source: DSMZ) from: Acholeplasma laidlawii, Mycoplasma pneumoniae, Mycoplasma arginini, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma fermentans, Spiroplasma citri.
- The kit meets EP 2.6.7 critreria and allows detection over 100 Mollicute species.
- Mycoplasma PCR Kit provides the efficient detection of Mollicutes with high sensitivity (LOD ≤ 10 CFU/ml or LOD ≤ 5 Mollicutes DNA copies per reaction).
- Mycoplasma Master Mix contains onTaq DNA Polymerase, optimized reaction buffer, dNTPs (dTTP is partially replaced dUTP), uracil-N-glycosylase (UNG), a gel loading reagent and two gel tracking dyes.
- onTaq DNA Polymerase is a modified “hot start” enzyme that is blocked at moderate temperatures and allows room temperature reaction setup.
- The polymerase activity is restored during the initial denaturation step when amplification reactions are heated at95°C for 15 minutes.
- Use of dUTP and uracil-N-glycosylase (UNG) prevents carryover contamination between reactions. UNG removes uracil from any dU-containing contaminating amplicons, leaving abasic sites and making DNA molecules susceptible to hydrolysis during the initial denaturation step.
- A gel loading reagent and two gel tracking dyes included in Mycoplasma PCR Kit allow PCR reactions to be loaded directly onto an agarose gel without prior addition of a gel loading buffer.
List of Mollicute strains detected with Duplex Mycoplasma qPCR Kit
M. agalactiae, M. agassizii, M. alkalescens, M. alligatoris, M. alvi, M. amphoriforme, M. anserisalpingitidis, M. arginini, M. arthritidis, M. bovigenitalium, M. bovirhinis, M. bovis, M. buccale, M. californicum, M. canadense, M. canis, M. capricolum, M. citelli, M. cloacale, M. collis, M. columbinum, M. columborale, M. conjunctivae, M. corogypsi, M. cottewii, M. cricetuli, M. crocodyli, M. cynos, M. dispar, M. edwardii, M. enhydrae, M. equirhinis, M. falconis, M. faucium, M. feliminutum, M. felis, M. fermentans, M. flocculare, M. gallisepticum, M. gateae, M. genitalium, M. gypis, M. hominis, M. hyopneumoniae, M. hyorhinis, M. hyosynoviae, M. imitans, M. iowae, M. leachii, M. lipofaciens, M. lipophilum, M. maculosa, M. microti, M. moatsii, M. molare, M. mucosicanis, M. muris, M. mustelae, M. mycoides, M. opalescens, M. orale, M. ovipneumoniae, M. oxoniensis, M. penetrans, M. phocae, M. phocicerebrale, M. phocidae, M. phocirhinis, M. phocoenae, M. phocoeninasale, M. pirum, M. pneumoniae, M. primatum, M. procyoni, M. pullorum, M. pulmonis, M. putrefaciens, M. salivarium, M. spermatophilum, M. spumans, M. sualvi, M. synoviae, M. testudineum, M. testudinis, M. timone, M. tullyi, M. venyonii, M. verecundum, M. volis, M. vulturii, M. yeatsii, M. zalophi
U. canigenitalium, U. diversum, U. gallorale, U. parvum, U. urealyticum,
S. apis, S. citri, S.ixodetis, S, kunkelli, S. poulsonii