Detection of mollicute contamination in cell cultures by 16S rDNA amplification.
A polymerase chain reaction (PCR) system was developed for the detection of mollicutes as contaminants of cell cultures. By using three oligonucleotides chosen in the 16S rDNA sequences, two sets of primers able to promote amplification of all Mycoplasma and Ureaplasma (molli1-molli2a) or all Acholeplasma (molli1-molli2b) species examined were determined. This PCR system, first applied to experimentally infected Vero cell lines, was then evaluated for the detection of mollicutes in 86 cell culture samples, comparatively to DNA staining, culture and ELISA. The results obtained by the four techniques were in agreement in 82 cases (36 positive, 46 negative). PCR allowed detection of contamination in one and two cases negative by ELISA and culture, respectively, and confirmed questionable results obtained by DNA staining. As described, PCR seems to be a very convenient tool for routine detection of cell culture contaminants.
Teyssou
R
R
Laboratoire de Bactériologie, Université de Bordeaux II, France.
Poutiers
F
F
Saillard
C
C
Grau
O
O
Laigret
F
F
Bové
J M
JM
Bébéar
C
C
D000127:Acholeplasma / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D004275:DNA, Ribosomal / Q000032:analysis* / Q000235:genetics; D005784:Gene Amplification; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000235:genetics / Q000302:isolation & purification; D009175:Mycoplasma Infections / Q000175:diagnosis; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D008973:Tenericutes / Q000235:genetics / Q000302:isolation & purification*; D014509:Ureaplasma / Q000235:genetics / Q000302:isolation & purification; D016869:Ureaplasma Infections / Q000175:diagnosis; D014709:Vero Cells
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D009841:Oligonucleotides