Comparison of 5 noncoding region primer sets in the detection of hepatitis C virus using reverse transcription polymerase chain reaction

Two new sets of nested primers were designed for the 5' noncoding region (NCR) of the hepatitis C virus (HCV). One hundred seventy HCV genome sequences were retrieved from GenBank and aligned. A consensus sequence was then determined for primer selection. A protocol was developed to test for the hepatitis C virus, after optimizing polymerase chain reaction (PCR) testing conditions for all four sets of nested primers. A comparison was performed between the two new sets of 5' NCR nested primers, WITTHCV1-4 and SKAHCV1-4, to two other 5' NCR primer sets, BUKHHCV1-4 and GRETHCV1-4, reported in the literature. One-hundred ninety-six serum specimens were used in the comparison. A majority of the specimens were submitted for Recombinant Immunoblot Assay 2 (RJBA) and blood bank donor testing. Other types of specimens, including RIB A indeterminate, hepatitis B, hepatitis A, HIV, and creatinine clearance, were also tested for possible cross-reactivity or interference. Reverse transcription polymerase chain reaction tests (RT-PCR) were performed using the four different nested primer sets. RT-PCR serum tests were performed with quality control measures in place. The purpose of comparing four nested primer sets of the 5' NCR was to determine if one set of nested primers may actually be more sensitive in the detection of HCV, thus reducing the proportion of false negatives. All four nested primer sets detected positive HCV RNA specimens equally; however, the two new primer sets WITT and SKA resulted in cleaner and easier to interpret band patterns than BUKH and GRET