Masukawa A, Miyachi H, Ohshima T, Fusegawa H, Ando Y
Central Clinical Laboratory, Tokai University Hospital, Isehara.
Rinsho Byori. 1997 Jul;45(7):673-8.
In the direct detection of pathogens by polymerase chain reaction (PCR) from clinical samples, false negative results due to the presence of inhibitor are problematic. In order to monitor such an inhibitor, we evaluated the detection of the positive internal control in PCR assay for HCV-RNA in serum samples positive for anti-HCV antibodies, of which 41 samples were positive for HCV-RNA by competitive RT-PCR assay. The positive internal control was coamplified with HCV-RNA and hybridized to the specific probe on magnetic beads and then hybrids were detected by colorimetric measurement using automatic PCR machine (COBAS AMPLICOR). Detection of the positive internal control was negatively correlated with viral copy number in sera assayed by competitive RT-PCR. Five of 52 samples (9.6%) with high HCV-RNA copy number (10(7) or 10(8) copies/ml) showed negative results for the internal control. The negative results for the internal control turned out to be positive when the sera were diluted and re-assayed, suggesting competitively inhibitory effects of high viral copy number on amplification of the internal control. Addition of heparin in the serum sample showed an inhibitory effect with a dose dependent manner on the detection of both HCV-RNA and the internal control, with a more effect on the lower copy number of HCV. On the other hand, addition of hemoglobin in the sample with concentration of up to 450mg/dl had no inhibitory effect on the detection of either HCV-RNA or the internal control. Coamplification and detection of the positive internal control was demonstrated to be useful to estimate effects of inhibitors, which may be present in clinical samples, in the detection of HCV-RNA by PCR.
