Detection of mycoplasma contaminations in cell cultures by PCR analysis.
Mycoplasma contamination is still one of the main problems in using cell cultures in biological and medical research and in the production of bioactive substances, because mycoplasma can alter nearly all parameters and products of the cell. They can persist undetected in the culture if no special detection methods are applied. In recent years, the PCR technology has become a commonly used method to analyze genomic DNA and the expression of genes, with both high specificity and sensitivity. This technique can be effectively employed for the detection and even the identification of mycoplasma contaminations in cell cultures applying primers complementary to the 16S rDNA region. Although this technique, once established, is characterized by simplicity and speed, PCR is still a complex process and its sensitivity and specificity can be influenced by a number of different parameters, e.g. inhibiting compounds originating from the preparation process of the DNA, RNA or cDNA, contamination of the solutions with PCR products, and the selection of a primer pair which does not cover all the mycoplasma species occurring in cell cultures. Thus, adequate controls have to be included to obtain reliable results. The present review examines the use of different primers of the 16S rDNA region including their specificity, the sensitivity applying various DNA or RNA preparation procedures, and the methods to detect finally the amplicons. In conclusion, basic nucleic acid preparation and PCR product detection methods offer a simple, fast and reliable technique for the examination of mycoplasma contaminations in cell cultures, provided that the indispensable control assays are implemented.
Uphoff
C C
CC
DSMZ-German Collection of Microorganisms & Cell Cultures, Department of Human and Animal Cell Cultures, Braunschweig, Germany. cup@dsmz.de
Drexler
H G
HG
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured / Q000382:microbiology; D003584:Cytological Techniques; D017931:DNA Primers; D006801:Humans; D009174:Mycoplasma / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers