Development of an amplification and hybridization assay for the specific and sensitive detection of Mycoplasma fermentansDNA

• Published in: Molecular and cellular probes Vol. 10; no. 1; pp. 7 - 14
• Main Authors: Berg, Silke, Lüneberg, Edeltraud, Frosch, Matthias
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 1996
• Subjects: DNA enzyme immuno assay; Mycoplasma fermentans; non-radioactive hybridization; polymerase chain reaction; species-specific oligonucleotides; species-specific oligonucleotides; DNA enzyme immuno assay; non-radioactive hybridization; Mycoplasma fermentans; polymerase chain reaction
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1006/mcpr.1996.0002

A polymerase-chain-reaction-based detection system for Mycoplasma fermentanswas established. The highly conserved tufgene, which encodes elongation factor Tu of prokaryotes, served as target sequence for the PCR. With two PCR oligodeoxynucleotides, which were selected from M. fermentansspecific sequences of the tufgene, we amplified a 850 base pair DNA fragment. Via the biotin-moiety of one primer the PCR fragments were immobilized on streptavidin-coated microtitre plates. After alkaline denaturation a digoxigenin-labelled M. fermentansspecific DNA probe was hybridized to the single stranded immobilized PCR fragment. Detection was performed by addition of an alkaline phosphatase conjugated anti-digoxigenin antibody. 4-methyl-umbelliferyl-phosphate was used as a fluorogenic substrate. Amplification of 10 fg chromosomal target DNA was detected by this «DNA enzyme immuno assay (DEIA)» technique, corresponding to seven genome copies. Our study supports the presumption that the tufgene proves to be a suitable target sequence for the PCR based detection of any bacterial species. Furthermore, hybridization of PCR fragments with radio-labelled DNA probes should no longer be necessary, because a very sensitive non-radioactive test system can easily be established with the «DEIA» technique.