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

• Published in: Molecular and cellular probes Vol. 10; no. 1; pp. 7 - 14
• Main Authors: Berg, S, Lüneberg, E, Frosch, M
• Format: Journal Article
• Language: English
• Published: England 01.02.1996
• Subjects: Base Sequence; DNA Probes; DNA, Bacterial - analysis; Enzyme-Linked Immunosorbent Assay - methods; Molecular Probe Techniques; Molecular Sequence Data; Mycoplasma fermentans - genetics; Mycoplasma fermentans - isolation & purification; Peptide Elongation Factor Tu - genetics; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Sequence Analysis, DNA; Species Specificity
• ISSN: 0890-8508
• DOI: 10.1006/mcpr.1996.0002

A polymerase-chain-reaction-based detection system for Mycoplasma fermentans was established. The highly conserved tuf gene, which encodes elongation factor Tu of prokaryotes, served as target sequence for the PCR. With two PCR oligodeoxynucleotides, which were selected from M. fermentans specific sequences of the tuf gene, 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. fermentans specific 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 tuf gene 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.