Ligase chain reaction (LCR) assay for semi-quantitative detection of HBV DNA in mononuclear leukocytes of patients with chronic hepatitis B.
A ligase chain reaction (LCR)-based approach to detect hepatitis B virus (HBV) DNA in peripheral blood mononuclear cells (PBMC) is described. Using this new amplification technique, we determined semi-quantitatively the amount of a short HBV S-gene fragment in PBMC lysates of 25 patients with different forms of chronic hepatitis (group A (n = 8), hepatitis B s antigen (HBsAg)+/hepatitis B e antigen (HBeAg)+; group B (n = 9), HBsAg+/HBeAg-; group C (n = 8), HBsAg-/HBeAg-). The LCR results were compared with the findings obtained with polymerase chain reaction (PCR) amplification of three distinct HBV gene regions (preS1/2, S and C) and related to the serological profiles of the patients. Depending on the primer pair used for PCR amplification, sensitivity of HBV LCR in PBMC was equivalent or slightly superior to PCR. The highest positivity rate for HBV DNA was observed in the HBeAg+ and HBV DNA seropositive group (8/8) and was lower in the other patient groups B (4/9) and C (1/8). Interestingly, HBV gene sequences could also be detected in the lymphocytes of an HBsAg negative patient and in two patients from group B who were both negative for serum viral particles by PCR. The rapid LCR procedure represents a reliable alternative to PCR for the sensitive detection of HBV DNA in PBMC samples. In combination with the automated IMx(TM)-system the new amplification technique may be routinely used for screening for HBV in whole blood samples and thus may help to better evaluate the risk of HBV reinfection in liver transplant recipients.
L Medizinische Klinik und Poliklinik, University of Mainz, Germany.
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D019411:Clinical Laboratory Techniques / Q000379:methods*; D004279:DNA, Viral / Q000302:isolation & purification* / Q000378:metabolism*; D006509:Hepatitis B / Q000175:diagnosis* / Q000235:genetics*; D006514:Hepatitis B Surface Antigens / Q000032:analysis; D006513:Hepatitis B e Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D008025:Ligases / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D008025:Ligases