Specificity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines
| Autor: | Manfred Wirth, Hans G. Drexler, Anne Hopert, Hansjörg Hauser, Cord C. Uphoff |
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| Rok vydání: | 1993 |
| Předmět: |
food.ingredient
Insecta Immunology Molecular Sequence Data Fluorescent Antibody Technique In Vitro Techniques medicine.disease_cause Polymerase Chain Reaction law.invention Microbiology Cell Line chemistry.chemical_compound Nucleic acid thermodynamics Mice food Mycoplasma law medicine Immunology and Allergy Agar Animals Humans DAPI Polymerase chain reaction biology Base Sequence Nucleic Acid Hybridization Ribosomal RNA biology.organism_classification Molecular biology chemistry Oligodeoxyribonucleotides Mollicutes Agarose |
| Zdroj: | Journal of immunological methods. 164(1) |
| ISSN: | 0022-1759 |
| Popis: | The polymerase chain reaction (PCR) amplification was used for the detection of mycoplasma contamination in 42 continuous cell lines. Using the microbiological cultivation on agar as the reference method, 29 cell lines were regarded as positive and 13 cell lines as negative. The double-step PCR analysis employed nested primers that anneal to gene sequences coding for the evolutionarily conserved 16 S rRNA of some 25 different mycoplasma species (including the ones most commonly found in cell cultures). In terms of the positivity or negativity of mycoplasma infection the results were identical for the agar assay and PCR amplification. All positive cell lines displayed distinct, unequivocal, objectively discernible bands on agarose gels while the non-infected specimens showed no DNA amplification. A simultaneously performed comparison with four other commonly used detection methods (DNA-RNA hybridization in solution, DAPI DNA fluorescence staining, immunostaining with a monoclonal antibody and an ELISA) showed that PCR produced significantly less false-negative or false-positive results than all the other methods. Furthermore, in dilution experiments, PCR correctly detected the infecting mycoplasmas at the lowest level of 1/10(4) whereas the other assays were less sensitive. It is concluded that double-step PCR employing nested primers is superior to other mycoplasma detection methods in many respects: simplicity and speed, high specificity and extreme sensitivity, objectivity and accuracy. |
| Databáze: | OpenAIRE |
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