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Development of a new duplex real-time polymerase chain reaction assay for hepatitis B viral DNA detection.

Sun S, Meng S, Zhang R, Zhang K, Wang L, Li J - Virol. J. (2011)

Bottom Line: Quantification of hepatitis B virus (HBV) DNA can be used for diagnosing HBV infection and monitoring the effect of antiviral therapy.The within-run precision coefficient of variation (CV) ranged from 1.02% to 2.73%, while the between-run CV ranged from 0.83% to 1.25%.It is suitable for high throughput screening and frequent HBV DNA level monitoring.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Center for Clinical Laboratories, Beijing Hospital, People's Republic of China.

ABSTRACT

Background: Quantification of hepatitis B virus (HBV) DNA can be used for diagnosing HBV infection and monitoring the effect of antiviral therapy. However, probably because of mismatches between the template and primer/probe, HBV DNA in some HBV infections could not be detected using currently available commercial assays with single primer/probe. By aligning the HBV sequences, we developed a duplex real-time polymerase chain reaction (PCR) assay using two sets of primers/probes and a specific armored DNA as internal control (IC).

Results: The limit of the duplex real-time PCR assay was 29.5 IU/ml, whereas the specificity was 100%. The within-run precision coefficient of variation (CV) ranged from 1.02% to 2.73%, while the between-run CV ranged from 0.83% to 1.25%. The optimal concentration of armored DNA IC in the HBV DNA duplex real-time PCR assay was 1 000 copies/ml. Data from 69 serum samples with HBV infection showed that the performance of the duplex real-time PCR assay was comparable to that of the COBAS Ampliprep/Cobas Taqman (CAP/CTM) HBV assay and was superior to those of the domestic commercial HBV assays.

Conclusions: The duplex real-time PCR assay is sufficiently sensitive, specific, accurate, reproducible and cost-effective for the detection of HBV DNA. It is suitable for high throughput screening and frequent HBV DNA level monitoring.

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Related in: MedlinePlus

Linearity of the duplex real-time PCR assay. Linearity of the duplex real-time PCR assay was determined using serial 10-fold dilutions of HBV standard in negative serum samples at the following concentrations: 10, 102, 103, 104, 105, and 106 IU/ml. Three replicates were tested in a single run at each concentration. Linear relationship between the Ct values and the log10 HBV DNA concentration in the samples at concentrations from 10 to 106 IU/ml yielded R = 0.993.
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Figure 1: Linearity of the duplex real-time PCR assay. Linearity of the duplex real-time PCR assay was determined using serial 10-fold dilutions of HBV standard in negative serum samples at the following concentrations: 10, 102, 103, 104, 105, and 106 IU/ml. Three replicates were tested in a single run at each concentration. Linear relationship between the Ct values and the log10 HBV DNA concentration in the samples at concentrations from 10 to 106 IU/ml yielded R = 0.993.

Mentions: Linearity of the duplex real-time PCR assay was determined using serial 10-fold dilutions of HBV standard in negative serum samples at the following concentrations: 10, 102, 103, 104, 105, and 106 IU/ml. Three replicates were tested in a single run at each concentration. The proportion of positive results obtained from each input concentration was subjected to probit regression analysis (Table 3). The LOD of the duplex real-time PCR assay was 29.5 IU/ml (95% confidence interval, 20.9-56.2 IU/ml). Linear regression analysis of the Ct values against the log10 HBV DNA concentration yielded R = 0.993 (Figure 1). The specificity of the duplex real-time PCR assay was 100% when testing HBV-negative serum samples.


Development of a new duplex real-time polymerase chain reaction assay for hepatitis B viral DNA detection.

Sun S, Meng S, Zhang R, Zhang K, Wang L, Li J - Virol. J. (2011)

Linearity of the duplex real-time PCR assay. Linearity of the duplex real-time PCR assay was determined using serial 10-fold dilutions of HBV standard in negative serum samples at the following concentrations: 10, 102, 103, 104, 105, and 106 IU/ml. Three replicates were tested in a single run at each concentration. Linear relationship between the Ct values and the log10 HBV DNA concentration in the samples at concentrations from 10 to 106 IU/ml yielded R = 0.993.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3116493&req=5

Figure 1: Linearity of the duplex real-time PCR assay. Linearity of the duplex real-time PCR assay was determined using serial 10-fold dilutions of HBV standard in negative serum samples at the following concentrations: 10, 102, 103, 104, 105, and 106 IU/ml. Three replicates were tested in a single run at each concentration. Linear relationship between the Ct values and the log10 HBV DNA concentration in the samples at concentrations from 10 to 106 IU/ml yielded R = 0.993.
Mentions: Linearity of the duplex real-time PCR assay was determined using serial 10-fold dilutions of HBV standard in negative serum samples at the following concentrations: 10, 102, 103, 104, 105, and 106 IU/ml. Three replicates were tested in a single run at each concentration. The proportion of positive results obtained from each input concentration was subjected to probit regression analysis (Table 3). The LOD of the duplex real-time PCR assay was 29.5 IU/ml (95% confidence interval, 20.9-56.2 IU/ml). Linear regression analysis of the Ct values against the log10 HBV DNA concentration yielded R = 0.993 (Figure 1). The specificity of the duplex real-time PCR assay was 100% when testing HBV-negative serum samples.

Bottom Line: Quantification of hepatitis B virus (HBV) DNA can be used for diagnosing HBV infection and monitoring the effect of antiviral therapy.The within-run precision coefficient of variation (CV) ranged from 1.02% to 2.73%, while the between-run CV ranged from 0.83% to 1.25%.It is suitable for high throughput screening and frequent HBV DNA level monitoring.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Center for Clinical Laboratories, Beijing Hospital, People's Republic of China.

ABSTRACT

Background: Quantification of hepatitis B virus (HBV) DNA can be used for diagnosing HBV infection and monitoring the effect of antiviral therapy. However, probably because of mismatches between the template and primer/probe, HBV DNA in some HBV infections could not be detected using currently available commercial assays with single primer/probe. By aligning the HBV sequences, we developed a duplex real-time polymerase chain reaction (PCR) assay using two sets of primers/probes and a specific armored DNA as internal control (IC).

Results: The limit of the duplex real-time PCR assay was 29.5 IU/ml, whereas the specificity was 100%. The within-run precision coefficient of variation (CV) ranged from 1.02% to 2.73%, while the between-run CV ranged from 0.83% to 1.25%. The optimal concentration of armored DNA IC in the HBV DNA duplex real-time PCR assay was 1 000 copies/ml. Data from 69 serum samples with HBV infection showed that the performance of the duplex real-time PCR assay was comparable to that of the COBAS Ampliprep/Cobas Taqman (CAP/CTM) HBV assay and was superior to those of the domestic commercial HBV assays.

Conclusions: The duplex real-time PCR assay is sufficiently sensitive, specific, accurate, reproducible and cost-effective for the detection of HBV DNA. It is suitable for high throughput screening and frequent HBV DNA level monitoring.

Show MeSH
Related in: MedlinePlus