Rapid detection of mycoplasma contamination in cell cultures by enzymatic detection of polymerase chain reaction (PCR) products.
Enzymatic detection of polymerase chain reaction (ED-PCR) was applied for rapid and easy identification of mycoplasmas from contaminated cell culture. This method was based on the capture of amplified products via biotin-streptavidine affinity and the detection of an incorporated hapten in amplified products with enzyme-linked antibody. Primers corresponding to common sequence of Mollicutes in 16S ribosomal RNA dominated gene was used. Nineteen of twenty Mollicutes so far reported as cell contaminants appeared positive by ED-PCR, whereas remaining one, Acholeplasma axanthum, appeared negative. Samples from sixty-two cell culture were tested for contamination of mycoplasmas by means of ED-PCR, cultivation, and electronmicroscopy. The results of ED-PCR were the same as those of cultivating method. The time required for all the detection process in ED-PCR was about 5 hr for 20 samples. We suggest that ED-PCR can be used in the rapid detection of mycoplasms from cell culture.
National Institute of Animal Health, Ibaraki, Japan.
H
H
Kobayashi
K
K
Yamamoto
M
M
Eguchi
M
M
Kubo
S
S
Nakagami
S
S
Wakisaka
M
M
Kaizuka
H
H
Ishii
D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D002460:Cell Line; D046508:Culture Techniques / Q000379:methods; D017931:DNA Primers; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D019809:Streptavidin
D001426:Bacterial Proteins; D017931:DNA Primers; D012336:RNA, Ribosomal, 16S; D001710:Biotin; D019809:Streptavidin