Clinical laboratory evaluation of a PCR assay for the detection of HCMV.
OBJECTIVE
In limited laboratory space and consonant with limited opportunities for contamination, to implement and incorporate into our diagnostic virology laboratory, a polymerase chain reaction assay for human cytomegalovirus detection with maximum sensitivity.


METHODS
Polymerase chain reaction, adapted for use with the enzyme uracil-n-glycosylase to avoid the potential for false positive reactions due to amplicon carryover was developed, optimized using two primer pairs, and performed on 361 specimens, i.e., body fluids and tissues submitted to the viral laboratory for detection of human cytomegalovirus. Polymerase chain reaction results were compared to shell vial assay.


METHODS
Louisiana State University Health Sciences Center, Shreveport LA.


METHODS
Using the shell vial assay as the reference, analytical sensitivity (lower limit of detection) as well as laboratory sensitivity and specificity of both primer pairs.


RESULTS
The lower limit of detection of our polymerase chain reaction assay was determined to be one focus-forming unit. Using the shell vial assay as the reference test, the sensitivity and specificity of both primer pairs were 96.5% and 94.3%, respectively. Polymerase chain reaction detected human cytomegalovirus in 6% of our culture-negative specimens.


CONCLUSIONS
Based upon this study, our recommendations include the following: 1) a housekeeping gene amplification control is required for diagnostic polymerase chain reaction; 2) a single primer pair can be utilized for clinical work without sacrificing too much sensitivity; and 3) the laboratory should maintain close contact with clinicians to discuss polymerase chain reaction interpretation.
Matthews-Greer
J M
JM
Diagnostic Virology Laboratory in Pediatrics, Louisiana State University Health Sciences Center at Shreveport, 71130-3932, USA. jgreer@lsumc.edu
McRae
K L
KL
Jamison
R M
RM
D019411:Clinical Laboratory Techniques; D003587:Cytomegalovirus / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D045647:DNA Glycosylases / Q000502:physiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D051981:Uracil-DNA Glycosidase; D014773:Virology / Q000379:methods
D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase