Comparison of sensitivity for human cytomegalovirus of the polymerase chain reaction, traditional tube culture and shell vial assay by sequential dilutions of infected cell lines.
Although traditional tube culture (TTC) is still considered by many as the 'gold standard' for the laboratory diagnosis of human cytomegalovirus (HCMV), the shell vial assay (SVA) offers greater speed of detection. This technique utilizes immunofluorescence (IF) to detect early or immediate early nuclear antigens (IEA). The detection capabilities of these two tests were compared with the polymerase chain reaction (PCR), a technique that amplifies enzymatically selected DNA target sequences. Serial dilutions of crude culture harvests from 2 HCMV strains, Towne and a clinical urine isolate, were made up to 1:1 000,000. Ten-microliters aliquots of the original sample and each dilution were tested by PCR, TTC and SVA. For PCR, the nested-primer approach was used. Outer primers delimited a 721-bp sequence contained within the 2nd to 4th exons of the immediate-early protein. Inner nest primers delimited a 167-bp sequence in the third exon, detected by a 32P-labelled probe. The results show that: (1) control samples which contained all PCR reagents but no DNA were uniformly negative; (2) radiolabelled-probe detection (RPD) of PCR products is, on average, 100 x more sensitive than detection by ethidium bromide; (3) PCR is, on average, 100 x more sensitive than evaluation of cytopathic effect (CPE) in the TTC; (4) the predictive value of a negative SVA result is low compared to PCR.
Sandin
R L
RL
George Washington University Medical Center, Department of Pathology, Washington, DC.
Rodriguez
E R
ER
Rosenberg
E
E
Porter-Jordan
K
K
Caparas
M
M
Nasim
S
S
Rockis
M
M
Keiser
J F
JF
Garrett
C T
CT
D001483:Base Sequence; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000382:microbiology; D004279:DNA, Viral; D005455:Fluorescent Antibody Technique; D006801:Humans; D007201:Indicator Dilution Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014776:Virus Cultivation
D004279:DNA, Viral