Use of the polymerase chain reaction to screen cell banks for retroviruses.
The polymerase chain reaction (PCR) may be used to increase the sensitivity for detecting nucleic acids of specific adventitious agents. PCR can be used in combination with other assays such as infectivity or co-cultivation to increase the sensitivity of detecting adventitious agents or to identify a particular adventitious agent present in the test sample. In addition, PCR is useful for confirming the results of other assays such as reverse transcriptase or electron microscopy. Sources of the test sample, passage history and raw materials added to the test sample are considered when deciding upon the appropriate screening procedures and the adventitious agents for which to screen. In developing a PCR assay, the experiments should be designed to address specificity, sensitivity and reproducibility. Assay development issues include design of primers for the particular retroviruses in question, determination of quantity of sample to test, potential levels of interference, and appropriate positive and negative controls. Validation of a PCR assay is necessary to prove reproducibility for detecting the retrovirus. Data are presented on the qualification of a primate master cell bank to assure the absence of simian immunodeficiency viruses.
Deatly
A M
AM
Lederle-Praxis Biologicals, Pearl River NY, USA.
McAuliffe
J M
JM
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line / Q000648:ultrastructure / Q000821:virology; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism; D004279:DNA, Viral / Q000302:isolation & purification; D015684:Gene Products, pol / Q000032:analysis; D008854:Microscopy, Electron; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000032:analysis; D015203:Reproducibility of Results; D015301:Retroviruses, Simian / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D015302:Simian Immunodeficiency Virus / Q000235:genetics / Q000302:isolation & purification; D013698:Templates, Genetic
D004279:DNA, Viral; D015684:Gene Products, pol; D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase