Standardization of a PCR-ELISA in serum samples: diagnosis of active parvovirus B19 infection.
To standardize a PCR assay for the detection of parvovirus B19 DNA in serum samples three different sample treatments were evaluated on the basis of the efficiency of recovery, reproducibility, convenience of sample handling, and presence of PCR inhibitors. Moreover, the presence of an internal standard competitor as the working reagent at one defined concentration in a competitive PCR-ELISA has been suggested as a valid tool to standardize and validate the assay. The results indicated that serum sample treatment by rapid heating fulfilled the criteria for a routine practice in the diagnostic laboratory. Titration experiments carried out to define the optimal amount of the internal standard competitor to use in PCR-ELISA showed that at 2 x10(2) competitor copies, any amplification interferences between target and competitor sequences were avoided. The internal standard competitor in a competitive PCR-ELISA allows the detection of false-negative results due to PCR inhibitors in the samples or large amounts of target DNA. Heating treatment and competitive PCR-ELISA for the detection of parvovirus B19 DNA were applied to the testing of 347 serum samples, which were submitted to the laboratory for B19 investigation. Of the 34 serum samples that were positive for B19 DNA, 15 were from adult patients and 19 from pediatric subjects. B19 infection was associated with haematological disorders, nonimmunological foetal hydrops, atypical rash, arthropathies, hepatic dysfunction, nonspecific symptoms, and congenital infections.
Zerbini
M
M
Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Bologna, Italy. mzerbini@med.unibo.it
Gallinella
G
G
Manaresi
E
E
Musiani
M
M
Gentilomi
G
G
Venturoli
S
S
D000293:Adolescent; D000328:Adult; D015415:Biomarkers / Q000097:blood; D002648:Child; D002675:Child, Preschool; D004279:DNA, Viral / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D006358:Hot Temperature; D006801:Humans; D010322:Parvoviridae Infections / Q000097:blood / Q000175:diagnosis* / Q000821:virology; D016732:Parvovirus B19, Human / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000592:standards*
D015415:Biomarkers; D004279:DNA, Viral