Construction of real-time RT-PCR using locked nucleic acid capture TaqMan probes and its detection of hepatitis B virus DNA

Objective To establish a novel system of real-time RT-PCR using locked nucleic acid (LNA) capture TaqMan probes for detection of hepatitis B virus (HBV) DNA. Methods Between August 2009 and March 2010, 20 healthy blood donors and 75 patients with COBAS Amplicor-identified HBV DNApositive chronic hepatitis B (CHB) in our Department of Hepatology received phlebotomy for specific assay with RT-PCR using LNA capture TaqMan probes. Meanwhile, the reproducibility, specificity and range of linearity of LNA RT-PCR were analyzed. Results The outcomes showed good linearity of LNA RT-PCR within the range of 10 ～ 2.3×109 IU/ml. A 95％ confidence interval (Cl) was identified with 10 IU/ml as the low limit. Linear regression analysis demonstrated a slope of 1.0 (R2=0.999), an intra-assay coefficient of variance ranging from 3.88％ to 4.36％, and an inter-assay coefficient of variance ranging from 8.5％ to 11.7％. The samples of control serum from 20 healthy blood donors were negative for HBV DNA, and all the samples without one from 75 patients were HBV DNA positive as detected by COBAS Amplicor.Conclusion RT-PCR using LNA capture TaqMan probes is a novel and quick test with high sensitivity and accuracy, which warrants its widespread use in clinical laboratory. Key words: Hepatitis virus, type B; Polymerase chain reaction (PCR) ; Nucleic acid probe; DNA, intergenic; Locked nucleic acid