Development of real-time PCR for detection of Mycoplasma hominis.
BACKGROUND
Mycoplasma hominis is associated with pelvic inflammatory disease, bacterial vaginosis, post partum fever, sepsis and infections of the central nervous system often leading to serious conditions. Association with development of female infertility has also been suggested, but different publications present different results. We developed a sensitive and fast diagnostic real-time PCR to test clinical samples from women undergoing laparoscopic examination before fertility treatment. To develop a test for the detection and quantification of M. hominis we selected a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gap), as a target.


RESULTS
Real-time PCR was optimized to detect 10 copies of M. hominis PG21 genomic DNA. A fluorescence signal was measured for all 20 other M. hominis isolates, and melting curves analysis showed variations in the melting temperature in agreement with sequence variation in the region of the probes. There was no amplification of other mycoplasmal DNA and human DNA. Eighty-three patient cervical swab samples from infertile women were cultured for M. hominis in the BEa medium. Two of the samples (2.4%) were positive after 48 hours of incubation. The real-time PCR detected the same two samples positive, and the DNA concentrations in the clinical specimens were calculated to 37.000 copies/ml and 88.500 copies/ml, respectively.


CONCLUSIONS
The results demonstrate that real-time PCR may prove to be a rapid alternative to the traditional cultivation method. Information on bacterial load in genital swabs can be obtained. The assay allowed detection of M. hominis in a closed system reducing the risk of contamination by amplicon carry-over.
Baczynska
Agata
A
Department of Medical Microbiology and Immunology, Aarhus University, Bartholin Building, 8000 Aarhus C, Denmark. agata@medmicro.au.dk
Svenstrup
Helle F
HF
Fedder
Jens
J
Birkelund
Svend
S
Christiansen
Gunna
G
D002584:Cervix Uteri / Q000378:metabolism / Q000382:microbiology; D003199:Computer Systems; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes; D004269:DNA, Bacterial; D019286:Endopeptidase K / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes; D005798:Genes, Bacterial / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D009175:Mycoplasma Infections / Q000175:diagnosis; D019535:Mycoplasma hominis / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014626:Vaginal Smears / Q000379:methods
D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D019286:Endopeptidase K