Quantitative Measurement of Hepatitis B Virus DNA by Competitive Nested Polymerase Chain Reaction / 대한임상병리학회지

ABSTRACT

BACKGROUND: We performed competitive nested polymerase chain reaction (PCR) to evaluate the clinical utility of quantitative measurement of HBV DNA by PCR and it's correlation with other serologic hepatits B markers. Because hepatitis markers such as HBsAg, HBeAg, anti-HBe can not accurately reflect the replication of hepatitis B virus (HBV). METHODS: The internal standard was generated from the HBV core gene by point mutation, which would result in restriction site for the restriction enzyme Eco RI and performed competitive nested PCR followed by densitometric scanning of the amplified products of agarose gel. RESULTS: The sensitivity of nested PCR was 5 molecules in direct observation of agarose gel, but because of the background effect as taking polaroid photo graph it was 50 molecules by using densitometer. When DNA pellets for original 250 microL serum were diluted with 40 microL distilled water the low detection limit was 5.0 x10(3) molecules/microL, however it could be lowered when less diluted. Lower detection limit of densitometer was 6.25 pg by twofold serial dilution of 100 pg of purified HBV DNA PCR products, and regression showed y=0.93x-0.33 (y density, x concentration, 6.25 pg considered as 6.25 density). The reproducibility of the densitometer from high concentration was 4.3 +/-0.6 x10(6) molecules/microL(mean +/-SD, CV 14%), and low concentration was 3.7 +/-0.7 x10(4) molecules/microL(mean +/-SD, CV 20%) Higher concentration of HBV DNA in HBeAg positive cases comparing with HBeAg negative cases was statistically significant (p<0.01). There was no correlation between HBV DNA concentration and serum value of alanine aminotransferase. CONCLUSION: Quantification of HBV DNA should be very useful in clinical follow-up of Post-therapy Patients and in anticipating Prognosis and infectivity of the disease, especially in cases of atypical hepatitis B and hepatitis B without seroconversion of routine hepatitis B markers. The shortcoming of the method seemed to be a rough estimate of HBV concentration as measuring the ratio of specimen/internal standard of two consecutive concentration among 10 folds serially diluted internal standard.