Development of a Molecular-Beacon Assay To Detect the G ₁₈₉₆ A Precore Mutation in Hepatitis B Virus-Infected Individuals

Abstract

The 1896 precore (PC) mutation is the most frequent cause of hepatitis B virus e-antigen (HBeAg)-negative chronic hepatitis B virus (HBV) infection. Detection of the 1896 PC mutation has application in studies monitoring antiviral therapy and the natural history of the disease. Identification of this mutation is usually performed by direct sequencing, which is both costly and laborious. The aim of this study was to develop a rapid, high-throughput assay to detect the 1896 PC mutation using real-time PCR and molecular-beacon technology. The assay was initially standardized on oligonucleotide targets and plasmids containing the wild-type (WT) and PC mutation and then tested on plasma samples from children with HBV DNA of >10 6 copies/ml. Nine individuals were HBeAg negative and suspected to harbor HBeAg mutations, while 12 children were HBeAg positive and selected as controls. Ninety percent (19 of 21) of plasma samples tested with molecular beacons were in complete agreement with sequencing results. The remaining 10% (2 of 21) of samples were identified as heterogeneous mixtures of WT and mutant virus by molecular beacons, though sequencing found only a homogeneous mutant in both cases. Overall, the 1896 PC mutation was detected by this assay in 55.5% of the children with HBeAg-negative infection. In summary, this assay is a rapid, sensitive, and specific technique that effectively discriminates WT from 1896 PC mutant HBV and may be useful in clinical and epidemiological studies.Approximately 350 million individuals are chronically infected with the hepatitis B virus (HBV) worldwide (14,20,21,23). HBV is the leading cause of liver cirrhosis and hepatocellular carcinoma (HCC) and is responsible for 60 to 80% of all HCC cases and more than 1 million deaths per year (20,33,34). The clinical course of HBV infection is monitored by biological markers in the serum, including liver enzymes such as alanine aminotransferase, and direct quantification of HBV DNA viral load. Seroconversion to HBV e-antigen (HBeAg)-negative and e-antibody (HBeAb)-positive status usually implies a decrease in viral replication, decrease in potential liver injury, improved prognosis, and decrease in HCC risk (16) and is the major end point of therapy (10,

Molecular Beacons: A Novel Optical Diagnostic Tool

“…It was detected using RT-PCR and molecular-beacon technology of the assay in 55.5 % of the children with HBeAg-negative infection (Sum et al 2004). The HBV genomic DNA level measured using RT-PCR with a MB (HBV beacon assay) showed good correlation with that measured by the commercially available COBAS AMPLI-COR HBV Monitor test (r = 0.901; p \ 0.001) (Waltz et al 2005). A sensitive and reproducible RT-PCR assay based on the universal MB (U-MB) technique was developed for the detection of HBV DNA in serum (Li et al 2007).…”

HBeAg-negative chronic hepatitis B: From obscurity to prominence

“…In most instances it runs a self-limited course [20] and only occasionally a fulminant one [21]. Infection with the ''wild'', HBeAg-producing strain, present in all HBeAgpositive and to a varying extent in most of HBeAg-negative chronic HBV carriers [22], is thought to be responsible for chronicity following an acute HBV infection [23]. The age at acute infection plays an important role in the development of chronicity.…”

Prevalence of the precore G1896A mutation in Chinese patients with e antigen negative hepatitis B virus infection and its relationship to pre-S1 antigen

“…Serum samples were collected and stored at -70ºC until use. The G1896A precore mutation was detected by a molecular-beacon assay described by Wartz et al (31). In Of the 496 patients, 380 (76.6%) had detectable serum HBV DNA by real-time PCR.…”