Usefulness of in-house PCR methods for hepatitis B virus DNA detection

Portilho, Moyra Machado; Baptista, Márcia L.; Silva, Messias da; Sousa, Paulo Sérgio Fonseca de; Lewis-Ximenez, Lia Laura; Lampe, Elisabeth; Villar, Lívia Melo

doi:10.1016/j.jviromet.2015.07.010

Cited by 4 publications

(2 citation statements)

References 20 publications

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“…The HBV DNA detection by in-house seminested PCR showed a concordance of 67.8% with the commercial technique. The authors thus concluded that the former technique showed an adequate concordance with some limitations, being a good method in low-resource settings (57).…”

Section: Discussionmentioning

confidence: 91%

Analysis of hepatitis B virus genotypes by restriction fragment length polymorphism

et al. 2015

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Section: Discussionmentioning

confidence: 91%

Analysis of hepatitis B virus genotypes by restriction fragment length polymorphism

et al. 2015

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“…The application of ultrasensitive HBV qPCR assays allows detection of viral breakthrough at an early stage as well as discontinuation of antiviral therapy at a proper time, thus improved the control of patients with low viral load [ 18 ]. However, the wide adoption of current ultrasensitive HBV quantitation assays in clinical laboratories faces challenges due to the time-consuming nature of the serum HBV DNA extraction as well as expensive costs of specialized equipment [ 21 ]. Therefore, developing a simple, rapid and cost-effective DNA extraction method is of particular importance for ultrasensitive HBV DNA detection.…”

Section: Introductionmentioning

confidence: 99%

Ultrasensitive detection of serum hepatitis B virus by coupling ultrafiltration DNA extraction with real-time PCR

Xiao

et al. 2017

PLoS ONE

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BackgroundA simple and reliable DNA extraction of hepatitis B virus (HBV) is critical in developing an ultrasensitive detection method for HBV infection. Current commercially available serum Hepatitis B Virus (HBV) DNA extraction methods are time-consuming, expensive and/or require specialized equipment, which hinders wide adoption of clinical laboratories. This study offers a report on an ultrasensitive HBV DNA detection method by coupling serum HBV DNA extraction by ultrafiltration (UF) with real-time PCR (qPCR) detection.MethodsSerum proteins were precipitated by phenol to release HBV DNA in the supernatant which was then transferred to the UF devices. The resultant DNA concentrate was eluted and released into qPCR pre-mixture. The UF-qPCR assay performance, including recovery rate, linearity, detection sensitivity, precision and diagnostic accuracy that compared to the CAP-CTM V2.0 assay by analyzing batched low viral load clinical samples was evaluated.ResultsThe recovery rate of the UF-based HBV DNA extraction method was above 80%. The assay linearity was demonstrated with a slope of 0.95 and R2 values of 0.99. Limit-of-detection (LOD) of the UF-qPCR assay was determined to be 12.1IU/ml. The coefficient of variation (CV) of HBV quantitation for high, low and limit titer samples was 2.28%, 5.77% and 25.59%, respectively. Accuracy of the UF-qPCR assay was confirmed with the reference panel, and there was a strong correlation between these two methods (R2 = 0.55, p < 0.01).ConclusionsThe UF-qPCR assay is reliable, highly sensitive, affordable and time-saving, and the method can be used for ultrasensitive detection of serum HBV.

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Dried blood spot sampling for hepatitis B virus quantification, sequencing and mutation detection

Bezerra

Portilho

Barbosa

et al. 2022

Sci Rep

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Hepatitis B virus (HBV) diagnosis is performed on serum samples, but the access to this diagnosis is difficult in low-income regions. The use of dried blood spot (DBS) samples does not require special structure for collection, storage or transport. This study evaluates the use of DBS for detection, quantification and sequencing of HBV DNA using in-house techniques. Two study groups were included: 92 HBsAg + individuals and 49 negative controls. Serum and DBS samples were submitted to quantitative and qualitative in-house PCR for S/pol genes, sequencing and phylogenetic analyses. Total of 84 serum samples were successfully amplified. Of them, 63 paired DBS were also positive in qualitative PCR. Qualitative PCR in DBS presented a sensitivity of 75% and specificity of 100% (Kappa = 0.689). Quantitative PCR in DBS presented a detection limit of 852.5 copies/mL (250 IU/mL), sensitivity of 77.63% and specificity of 100% (Kappa = 0.731). A total of 63 serum samples and 36 DBS samples were submitted to sequencing, revealing the circulation of genotypes A (65.08%), D (4.8%), E (3.2%) and F (27%) with 100% of correspondence between serum and DBS. All sequenced samples displayed polymorphisms in HBsAg gene. An HIV-coinfected patient presented the rtM204V/I-rtL180M double resistance mutation in serum and DBS. In conclusion, DBS is an alternative to detect, quantify and characterize HBV DNA, being a possibility of increasing diagnosis in low-income settings, closing gaps in HBV control.

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Usefulness of in-house PCR methods for hepatitis B virus DNA detection

Cited by 4 publications

References 20 publications

Analysis of hepatitis B virus genotypes by restriction fragment length polymorphism

Analysis of hepatitis B virus genotypes by restriction fragment length polymorphism

Ultrasensitive detection of serum hepatitis B virus by coupling ultrafiltration DNA extraction with real-time PCR

Dried blood spot sampling for hepatitis B virus quantification, sequencing and mutation detection

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