COMPARISON OF DOT-BLOT HYBRIDIZATION, HYBRID CAPTURE AND POLYMERASE CHAIN REACTION IN THE DETECTION AND QUANTITATION OF HEPATITIS B VIRUS DNA IN SERUM
HO SKN, CHEUNG IKP, CHAN TM.
Division of Nephrology, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong
Hepatitis B virus (HBV) infection can lead to life-threatening complications such as cirrhosis or fulminant hepatic failure, particularly in immunosuppressed patients. In contrast to the level of liver enzymes, which reflect the degree of hepatic inflammation, detection of HBV DNA in serum provides direct evidence for viral replication. Furthermore, recent data suggest that serial monitoring of HBV DNA level can be helpful in clinical management. We compared the performance of the dot-blot hybridization (DB), the Digene hybrid capture (HC), and the nested polymerase chain reaction (PCR) assays for the measurement of serum HBV DNA.
Our in-house non-radioisotope DB assay yields semi-quantitative results within 2 days, with a sensitivity of 100 pg/ml, which is comparable to conventional radioactive DB assays. The HC assay, which measures the level of HBV DNA by hybridization of RNA probes to single-stranded DNA and antibody capture of hybrids, followed by chemiluminescence, has a sensitivity of 10 pg/ml. Primer sets for the surface antigen and core antigen coding regions are utilized for the nested PCR assay. Our PCR assay has a sensitivity of 0.01 fg/ml (3 copies of HBV genome per assay). Detection of HBV DNA in 121 serum samples from 20 HBsAg+ renal transplant recipients by DB, HC and PCR assays yielded positivity rates of 57%, 74% and 100% respectively. None of 20 serum samples from 12 HBsAg- individuals yielded positive results. All the three assays had 100% specificity. The DB assay was more prone to inter-assay variation in view of its semi-quantitative nature. There was no correlation between HBV DNA and ALT levels.
We conclude that while the PCR assay provides a most sensitive means to detect serum HBV DNA, the HC assay may be more useful for the serial monitoring of patients, especially those treated with anti-viral agents.
Copyright 1997 Hong Kong Medical Technology Association .
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