[Clinical evaluation of RT-PCR method for detection of HCV-RNA: second report availability of RT-PCR system using PCR internal control for detection of HCV-RNA and the influence of interfering substances on the detection].
The COBAS Amplicor system is an automated diagnostic PCR system which contains a PCR internal control (P. I. C.) template to monitor the amplification. The applicability of COBAS Amplicor HCV was examined using sera of patients with hepatitis. Furthermore, the effects of possible interfering substance (total protein, triglyceride, hemoglobin, glucose, total bilirubin, heparin, lysis reagent including guanidium) on HCV-RNA detection were investigated. The sensitivity of COBAS Amplicor HCV was equivalent to the manual method of Amplicor HCV, moreover all of the results in 54 clinical samples analyzed on both COBAS Amplicor HCV and Amplicor HCV were in agreement. Detection sensitivity of HCV-RNA decreased in the presence of total bilirubin and heparin. Ten and 25mg/dl of total bilirubin affected HCV-RNA detection but did not affect P. I. C. This result suggested that total bilirubin interfered with the protein denature caused by the lysis reagent. Fifteen U/ml of heparin in the sample completely inhibited amplification both of the HCV-RNA and P. I. C. One U/ml of heparin did not affect amplification, but heparinized blood samples should not be used for the detection of HCV-RNA. To examine the effect of possible carry over contamination on the lysis reagent which contains guanidium, various concentrations of lysis reagent in P. I. C. were tested. RT-PCR was inhibited by 1/500 volume contamination of lysis reagent in specimen diluent. Other substances did not affect the sensitivity. Our results indicate that the carryover contamination of lysis reagent cause more "false negative" results than interfering substances in sera. In conclusion, HCV-RNA detection system containing P. I. C., such as COBAS Amplicor HCV, will become a very useful to differentiate "false negative" and "true negative" result.
Furuwatari
C
C
Central Laboratories, Shinshu University Hospital, Matsumoto.
Hidaka
E
E
Akahane
M
M
Ishikawa
M
M
Ueno
I
I
Furihata
K
K
Ogiso
Y
Y
Katsuyama
T
T
D001663:Bilirubin; D016174:Hepacivirus / Q000235:genetics*; D006493:Heparin; D006801:Humans; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D011237:Predictive Value of Tests; D012367:RNA, Viral / Q000032:analysis*; D012680:Sensitivity and Specificity
D012367:RNA, Viral; D006493:Heparin; D001663:Bilirubin