Application of PCR for detection and identification of mycoplasma contamination in virus stocks.
A nested polymerase chain reaction (PCR) was used to detect and identify mycoplasma contaminants in viral stocks. The results of the PCR assay proved to be a sensitive and accurate indicator of the true status of the stock tested. Those samples positive by agar culture or Hoechst stain were also positive by PCR. Those samples that were inconclusive by Hoechst stain (10.05%) could be clearly determined to be mycoplasma positive or negative by PCR. The PCR assay also detected those fastidious species of mycoplasma that gave false negative results by the direct culture method. In many respects the PCR-based mycoplasma detection method described is superior to the agar culture and Hoechst staining detection methods. In this study, the PCR assay detected substantially more mycoplasma-positive viral stocks than did the agar culture assay. Due to its speed, sensitivity, and reliability, the PCR assay is of particular value in monitoring the process of removing mycoplasma from contaminated stocks. Furthermore, the PCR amplification products can be analyzed by restriction analysis to rapidly identify the species of the mycoplasma contaminating the stock tested.
Hu
M
M
Department of Virology, American Type Culture Collection, Rockville, Maryland 20852, USA.
Buck
C
C
Jacobs
D
D
Paulino
G
G
Khouri
H
H
D001483:Base Sequence; D006690:Bisbenzimidazole / Q000737:chemistry; D004396:Coloring Agents / Q000737:chemistry; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D014780:Viruses / Q000302:isolation & purification*
D004396:Coloring Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D006690:Bisbenzimidazole