AMPLIFICATION AND DETECTION OF THE TERMINAL 3'-NONCODING REGION OF HEPATITIS-C VIRUS ISOLATES

A reverse transcription polymerase chain reaction (RT-PCR) assay was s et up to amplify, from chronically infected patients, the recently dis covered hepatitis C virus (HCV) 3' non-coding region (3'NCR). A panel of 149 samples was tested by RT-PCR for the 3'NCR. Two detection metho ds of amplified products were evaluated: ethidium bromide staining on 3% agarose gel electrophoresis and DNA enzyme immunoassay (''DEIA''), Results were compared with those obtained by amplification of the 5' n oncoding region (5'NCR), i.e. the ''Amplicor'' HCV RNA qualitative ass ay. Genotype distribution of the 86 Amplicor-positive samples was subt ype la: n=15 (17.4%); subtype Ib: n=32 (37.2%); subtype 2a/2c: n=7 (8. 1%); type 3: n=25 (29%); type 4: n=2 (2.3%); type 5: n=1 (1.2%); not d etermined: n=4 (2.3%). Sixty-three sera were HCV RNA-Amplicor-negative , 32 of which were from HCV-seronegative patients and 31 from HCV-sero positive patients, All seronegative samples were negative by both PCR methods. None of the Amplicor-negative samples from seropositive patie nts were positive by the 3'NCR assay. Forty-seven (54.7%) and 83 (96.5 %) of the 86 Amplicor-HCV-RNA-positive samples were positive after eth idium bromide staining and by the 3'NCR assay using DEIA, respectively . The limit of detection by end-point dilution was lower with Amplicor . No difference between genotypes was detected for the 3'NCR RT-PCR, a nd a high degree of concordance was obtained between the Amplicor and the 3'NCR DEIA results (97.4%). Nevertheless, further studies are need ed before the 3'NCR RT-PCR assay could be used instead of the 5'NCR RT -PCR for diagnostic purposes.