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 la rgely depend on viral DNA detection by polymerase chain reaction (PCR) or radioimmunological assay of viral antigens or antibodies. The qual ity 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 at tempted to improve the conditions of current PCR methods for detection of HBV DNA. In the present study, we applied a recently developed met hod of releasing HBV DNA from virion by NaOH treatment of patient seru m. Using four different primer sets specific to the HBV core region, w e found that the sensitivity of first-round PCR can be improved by mor e than two orders of magnitude depending on the primers. The second ro und of PCR using nested primers was sensitive enough to detect up to 1 0(-6) pg of the HBV DNA, which is equivalent to approximately 3 copies of the HBV genome. Among the approximately 800 HBV-infected patient s era investigated in our laboratory, more than 60% of the tested sample s gave positive results in the first-round PCR. The rate of positive r esults obtained using our experimental conditions is very high in comp arison with other reports. The reamplification of the first-round PCR reaction mixture with the nested primers produced practically 100% pos itive results. For diagnosis of HBV infection, we routinely used 1 mu l of patient serum, which was found to be optimum in our laboratory. S urprisingly, from 20% of our positive results, even serum diluted to 1 /100 (0.01 mu l) produced a stronger signal than 1 mu l. This observat ion suggests that direct PCR amplification of HBV DNA released from se rum by NaOH treatment has to be compensated by other DNA detection met hods for correct quantitation. In order to eliminate the false positiv e signal resulting from the carry-over due to massive screening of a l arge number of samples, PCR reaction mixture containing 8-methoxypsora len was exposed to ultraviolet light prior to thermal cycle amplificat ion. This exercise did not decrease the sensitivity of the detection m ethod, but almost completely removed the false positive results caused by contaminated templates. We are in the process of improving PCR-med iated HBV DNA detection methods to attain more reliable and easily app licable methods.