Internal controls as performance monitors and quantitative standards in the detection by polymerase chain reaction of herpes simplex virus and cytomegalovirus in clinical specimens.
Internal controls (IC) were produced and characterized for an HSV and a CMV PCR assay which serve both as test performance monitors and as quantitative standards. In each of the PCR assays the IC and native targets were amplified with equal efficiency and were detected with the same sensitivity, i.e. < 10 target copies. An algorithm was developed for the use of IC as a quantitative standard which entailed coamplifying a test specimen with four two-fold dilutions of the respective IC target (63-500 copies), followed by regression analysis of the relative yield of amplification products. This approach allowed the determination of both the initial virus genome copy number and the variability of the results, which provided a confidence index for the PCR assay. The relative yields of PCR products were determined by Southern blot and probe hybridization and by densitometry of digitized ethidium bromide-stained gels. Both methods produced estimations of the initial target copy numbers within +/- 40% of the expected value. Such a comprehensive analysis of an internal control for a PCR assay provides a rigorous control of test performance and permits reliable quantitative interpretation of a PCR assay result.
Nash
K A
KA
Department of Pathology, Childrens Hospital Los Angeles, CA 90027, USA.
Klein
J S
JS
Inderlied
C B
CB
D017088:AIDS-Related Opportunistic Infections / Q000175:diagnosis; D000293:Adolescent; D000465:Algorithms; D001483:Base Sequence; D002648:Child; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D006561:Herpes Simplex / Q000175:diagnosis; D018258:Herpesvirus 2, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D004279:DNA, Viral; D015345:Oligonucleotide Probes