"Single step" PCR with a sensitivity similar to nested PCR for the detection of hepatitis C virus RNA.
OBJECTIVE
Evaluation of the performance of different HCV PCR detection systems for HCV RNA: A nested PCR, considered the reference assay, was compared with two single-step methods (ss-PCR): the first is based on the detection of PCR products by liquid hybridization with a 32P end-labelled probe (isotopic ss-PCR), while the second assay is a colorimetric method (colorimetric ss-PCR) using microwell plate hybridization with a specific nucleic acid probe (Amplicor HCV PCR, Roche Diagnostics Systems).


METHODS
Sera from 56 patients with suspected hepatitis C infection based on reactive serology or altered liver parameters, and sera from 15 blood donors were tested for HCV RNA: After RNA extraction, the synthesized HCV cDNA was amplified in parallel using isotopic ss-PCR, colorimetric ss-PCR and nested PCR. The products were detected by autoradiography, color development and ethidium bromide fluorescence, respectively.


RESULTS
In order to assess the analytical sensitivity of ss-PCR versus that of nested of PCR, experiments included serial dilutions of positive control samples. Results showed that both methods had an extinction signal at the 1:512 dilution. A comparative analysis of 71 clinical sera samples was obtained using the three protocols and the results clearly documented 100% concordance.


CONCLUSIONS
Single step PCR methods for HCV RNA have a sensitivity equal to that of nested PCR and appear more suitable for diagnostic applications. Ss-PCR is safer than nested PCR in terms of both specificity and contamination problems. In particular, the Roche Amplicor HCV PCR assay minimizes sample exposure and management problems.
Farma
E
E
Laboratory of Virology, AIDS Center S. Luigi, Hospital S. Raffaele, Milan, Italy.
Boeri
E
E
Morsica
G
G
McDermott
J
J
Soldini
L
L
Repetto
C M
CM
Ferioli
B
B
Pellizzari
G
G
Molinari
F
F
Molinelli
A
A
D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000097:blood / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D012680:Sensitivity and Specificity
D012367:RNA, Viral