Real-Time PCR Assay for Detection and Quantification of Hepatitis B Virus Genotypes A to G

Welzel, Tania M.; Miley, Wendell; Parks, Thomas; Goedert, James J.; Whitby, Denise; Ortiz-Conde, Betty A.

doi:10.1128/jcm.00024-06

Cited by 56 publications

(36 citation statements)

References 34 publications

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“…Comparison of the two assays for quantitative detection of HBV DNA levels in the 118 serum samples showed that the two sets of results were highly correlated (r = 0.948, P < 0.001). This was in accordance with the results of previous studies that have evaluated other realtime PCR assays for quantitation of HBV DNA [13,14,[22][23][24][25][26] .…”

Section: Discussionsupporting

confidence: 92%

Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

Shi¹,

Zhang²,

Zhu³

et al. 2008

WJG

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INTRODUCTIONIt is estimated that 350 million individuals are chronically infected with hepatitis B virus (HBV) worldwide, and about one-third of these live in China [1] . HBV infection can cause chronic hepatitis, cirrhosis and hepatocellular carcinoma [2] . Among patients with active viral replication, cirrhosis will develop in 15%-20% within 5 year [3] , and 70-90 percent of cases of hepatocellular carcinoma occur against a background of cirrhosis [4] . Quantitation of HBV DNA in serum or plasma has been an important tool in identifying individuals with active hepatitis B, to monitor the efficiency of antiviral treatment, and to predict whether the treatment will be successful [5][6][7][8][9] .Several commercial molecular assays have been developed for quantitation of HBV DNA in serum or plasma samples. One of these assays is the COBAS Amplicor HBV Monitor, which is based on the amplification of DNA targets by the use of HBV-specific primers. Other methods, such as the VERSANT HBV DNA 3.0 assay, which is based on branched-DNA signal amplification, and Hybrid Capture Ⅱ, which is based on hybridization of a chemiluminescent probe, are also routinely used in diagnostic laboratories [10,11] . However, the costs of these assays are too high to be used in developing countries such as China, and in these countries the HBV burden is much heavier than in developed countries.Recently, quantitative real-time PCR has been used to detect and quantify HBV levels in serum or plasma samples. This assay is based on the relationship between the initial DNA target levels and the threshold cycle (Ct) of the amplification products. The Ct value is smaller when the initial DNA target level is higher. Several evaluation studies have shown that real-time PCR has a higher sensitivity, a broader dynamic range, and an accurate quantitation of HBV DNA compared to those of the existing commercial methods [12][13][14][15] .The Fosun real-time PCR HBV kit is a commercial METHODS:The reference sera of the Chinese National Institute for the Control of Pharmaceutical and Biological Products and the National Center for Clinical Laboratories of China, and 158 clinical serum samples were used in this study. The linearity, accuracy, reproducibility, assay time, and costs of the real-time PCR were evaluated and compared with those of the Cobas Amplicor test. RESULTS:The intra-assay and inter-assay variations of the real-time PCR ranged from 0.3% to 3.8% and 1.4% to 8.1%, respectively. The HBV DNA levels measured by the real-time PCR correlated very well with those obtained with the COBAS Amplicor test (r = 0.948). The real-time PCR HBV DNA kit was much cheaper and had a wider dynamic range. CONCLUSION:The real-time PCR assay is an excellent tool for monitoring of HBV DNA levels in patients with chronic hepatitis B.

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Section: Discussionsupporting

confidence: 92%

Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

Shi¹,

Zhang²,

Zhu³

et al. 2008

WJG

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“…Several recent articles compared results of HBV DNA quantification between the Versant branched DNA (bDNA) assay, using hybridization with multiple regions of the genome and presumably detecting both spliced and unspliced DNA (total DNA), and other quantitative assays using primers from the S region (Abbott RealTime HBV DNA and an in-house assay) (24,38) or the Roche Cobas TaqMan HBV DNA quantitative assay assumed to use S primers (detect only unspliced HBV DNA) (2,11,25) and another in-house assay using X region primers detecting both spliced and unspliced total HBV DNA (37). For the Abbott assay, the viral load was overall lower than it was with the bDNA assay but more so in 26% of sample pairs of genotypes C and A but not E (24) and lower by 0.67 log in Taiwanese samples mostly of genotype B (38).…”

Section: Discussionmentioning

confidence: 99%

Hepatitis B Virus DNA Splicing in Lebanese Blood Donors and Genotype A to E Strains: Implications for Hepatitis B Virus DNA Quantification and Infectivity

2012

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Hepatitis B virus (HBV) is one of the major viruses transmissible by blood that causes chronic infection in immunocompromised individuals. The study of 61 HBV carrier blood donors from Lebanon revealed multiple patterns of spliced HBV DNA. HBV DNA splicing was examined and quantified in samples of five genotypes and in seroconversion panels. The Lebanese sample median viral load was 1.5 ×10 2 IU/ml. All strains were genotype D, serotype ayw; 35 clustered as subgenotype D1 and 7 clustered as subgenotype D2. Three splice variants (SP1, SP1A, and Pol/S) were observed in 12 high-viral-load samples. Twenty samples of each genotype, A to E, were tested for the presence of HBV spliced DNA and SP1-specific splice variant. An unspliced HBV genome was dominant, but 100% of strains with a viral load of ≥10 5 copies/ml contained various proportions of spliced DNA. SP1 was detected in 56/100 (56%) samples in levels that correlated with the overall viral load. HBV DNA quantification with S (unspliced) and X (total DNA) regions provided different levels of viral load, with the difference corresponding to spliced DNA. During the highly infectious window period, the SP1 variant became detectable shortly after the hepatitis B surface antigen (HBsAg), suggesting a correlation between the initiation of splicing and the production of detectable levels of HBsAg. The quantification of HBV DNA with primers located outside and inside the spliced region might provide different estimations of viral load and differentiate between infectious and defective viral genomes. The role of splicing neoproteins in HBV replication and interaction with the host remains to be determined.

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“…Three different hairpin probes (probe-HBV-1, probe-HBV-2and probe-HBV-3) and corresponding primers were designed against the different conserved regions of the HBV genome including overlapping genes encoding X-protein, S gene and X gene region [2,21,22] (Table S2, S3 & S4). Under the optimized PCR amplification conditions of corresponding probes, the different colorimetric results by using those three pairs of probes were turned out (Fig.…”

Section: Evaluation and Selection Of Different Hbv Probesmentioning

confidence: 99%

A Novel Colorimetric PCR-Based Biosensor for Detection and Quantification of Hepatitis B Virus

Yang

Mei

et al. 2017

Biosensors and Biodetection

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We have established an operationconvenient and cost-effective way for detection HBV. This method has shown good performance with a broad range of linearity and high sensitivity. The results of detection is reported with macroscopic colorimetric signals. The strategy takes the key advantage of the TaqMan technology and use inexpensive DNA sensor. This approach can detect HBV DNA qualitatively or quantificationally. Hepatitis B virus (HBV) can cause viral infection that attacks the liver and it is a major global health problem that put people at a high risk of death from cirrhosis of the liver and liver cancer. HBV has infected one third of the worldwide population, and 350 million people suffer from chronic HBV infection. For these reasons, development of an accurate, sensitive and expedient detection method for diagnosing, monitoring and assessing therapeutic response of HBV is very necessary and urgent for public health and disease control. Here we report a new strategy for detection of viral load quantitation of HBV based on colorimetric polymerase chain reaction (PCR) with DNAzyme-containing probe. The special DNAzyme adopting a G-quadruplex structure exhibited peroxidase-like activity in the presence of hemin to report colorimetric signal. This method has shown a broad range of linearity and high sensitivity. This study builds important foundation to achieve the specific and accurate detection level of HBV DNA with a low-cost and effective method in helping diagnosing, preventing and protecting human health form HBV generally all over the world and especially in developing countries.

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Real-Time PCR Assay for Detection and Quantification of Hepatitis B Virus Genotypes A to G

Cited by 56 publications

References 34 publications

Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

Hepatitis B Virus DNA Splicing in Lebanese Blood Donors and Genotype A to E Strains: Implications for Hepatitis B Virus DNA Quantification and Infectivity

A Novel Colorimetric PCR-Based Biosensor for Detection and Quantification of Hepatitis B Virus

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