Primers determine the sensitivity of PCR-mediated hepatitis B virus DNA detection and pretreatment of PCR mixture with 8-methoxypsoralen eliminates false-positive results.
Most methods for the diagnosis of hepatitis B virus (HBV) infection largely depend on viral DNA detection by polymerase chain reaction (PCR) or radioimmunological assay of viral antigens or antibodies. The quality assurance program recently established in Europe reported that PCR-mediated HBV DNA detection methods used in many laboratories produced a high rate of false-positive and false-negative results. Thus, we attempted to improve the conditions of current PCR methods for detection of HBV DNA. In the present study, we applied a recently developed method of releasing HBV DNA from virion by NaOH treatment of patient serum. Using four different primer sets specific to the HBV core region, we found that the sensitivity of first-round PCR can be improved by more than two orders of magnitude depending on the primers. The second round of PCR using nested primers was sensitive enough to detect up to 10(-6) pg of the HBV DNA, which is equivalent to approximately 3 copies of the HBV genome. Among the approximately 800 HBV-infected patient sera investigated in our laboratory, more than 60% of the tested samples gave positive results in the first-round PCR. The rate of positive results obtained using our experimental conditions is very high in comparison with other reports. The reamplification of the first-round PCR reaction mixture with the nested primers produced practically 100% positive results. For diagnosis of HBV infection, we routinely used 1 microliter of patient serum, which was found to be optimum in our laboratory. Surprisingly, from 20% of our positive results, even serum diluted to 1/100 (0.01 microliter) produced a stronger signal than 1 microliter. This observation suggests that direct PCR amplification of HBV DNA released from serum by NaOH treatment has to be compensated by other DNA detection methods for correct quantitation. In order to eliminate the false positive signal resulting from the carry-over due to massive screening of a large number of samples, PCR reaction mixture containing 8-methoxypsoralen was exposed to ultraviolet light prior to thermal cycle amplification. This exercise did not decrease the sensitivity of the detection method, but almost completely removed the false positive results caused by contaminated templates. We are in the process of improving PCR-mediated HBV DNA detection methods to attain more reliable and easily applicable methods.
- 한국분자·세포생물학회 : 저널
- 원문이 없습니다.
원문복사신청을 하시면, 일부 해외 인쇄학술지의 경우 외국학술지지원센터(FRIC)에서
무료 원문복사 서비스를 제공합니다.
NDSL에서는 해당 원문을 복사서비스하고 있습니다. 위의 원문복사신청 또는 장바구니 담기를 통하여 원문복사서비스 이용이 가능합니다.
- 이 논문과 함께 출판된 논문 + 더보기