Quantitative Detection of Hepatitis B Virus DNA with a New PCR Assay

Kessler, Harald H.; Pierer, Karen; Santner, Brigitte I.; Vellimedu, Srinivas K.; Stelzl, Evelyn; Lackner, Herwig; Moser, Andrea; Marth, Egon

doi:10.1515/cclm.1998.105

Cited by 6 publications

(18 citation statements)

References 28 publications

(22 reference statements)

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“…The solvents were then removed in vacuo, and the conjugate was resuspended in 200 L of water. Conjugate [1] was used as described for ELOSA assays.…”

Section: Synthesis Of Poly(ma/mve)-odn Conjugate [1]mentioning

confidence: 99%

“…For conjugate [1] [obtained by coupling the 5Ј amino linker ODN1 to poly(MA/MVE)], the coupling yield was estimated as 88% from the SEC chromatogram, which corresponded to 13 ODNs bound to the chain.…”

Section: Estimation Of Odn Number Per Polymer Chainmentioning

confidence: 99%

“…The target used was a PCR product of HBV DNA (double-stranded 2339 bp) cloned in pBR 322 and purified by agarose gel electrophoresis, as previously described by Erout et al 9 The test was based on a sandwich hybridization system 16,17 with an additional step with 17 specific probes complementary to highly conserved regions of the HBV DNA sequence. 10 At the capture phase, conjugate [1] or [2] was coated (1 h at 37°C) on the inside of a disposable conical polystyrene-polybutadiene solid-phase receptacle (SPR) by passive adsorption. 10 The conjugate coating concentration (corresponding to the concentration of ODNs bound to the polymer chain) was 150 nM in phosphate buffered saline (PBS).…”

Section: Detection Of Hbv Dna By the Elosa Testmentioning

confidence: 99%

“…A poly(MA/MVE)-ODN conjugate [1] was used at the capture phase (Fig. 9) because it was described as the best candidate for amplifying the signal of HBV diagnostic tests run on the VIDAS instrument.…”

Section: Assessment Of the Various Conjugate Performances In An Elosamentioning

confidence: 99%

“…However, this very sensitive method often lacks reproducibility and requires stringent conditions to avoid contamination. 1 As another approach, the use of branched DNA (bDNA) has also been described with the aim of the direct detection of DNA [2][3][4] without enzymatic amplification. This strategy affords an accurate and quantitative signal, 5,6 and intensive investigations on the reduction of nonspecific hybridization have efficiently improved the sensitivity.…”

Section: Introductionmentioning

confidence: 99%

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Oligonucleotide synthesis onto poly(N‐acryloylmorpholine‐co‐N‐acryloxysuccinimide): Assessment of the resulting conjugates in a DNA sandwich hybridization test

Minard‐Basquin

Chaix

D’Agosto

et al. 2004

J of Applied Polymer Sci

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ABSTRACT:A water-soluble statistical poly(N-acryloylmorpholine-co-N-acryloxysuccinimide) [poly(NAM/NAS)] copolymer was studied for polymer-oligonucleotide (ODN) conjugate elaboration and for further use in diagnostic applications. Three different copolymers were first prepared by free-radical solution polymerization with different Nacryloylmorpholine (NAM) and N-acryloxysuccinimide (NAS) molar ratios (80/20, 70/30, and 60/40). Their number-average molecular weights ranged from 98,000 to 120,000 g/mol, as determined by aqueous size exclusion chromatography with an online light-scattering detector. Then, polymer-ODN conjugates were obtained via a strategy consisting of the direct synthesis of ODNs onto polymer chains previously grafted onto a controlled pore glass support. Before the grafting of the polymer onto the solid support, a preliminary step was performed to bind a nucleotide starter along the polymer chain (via the reactive NAS units) to initiate automated DNA synthesis. To multiply the number of ODNs growing from starters, a branched phosphoramidite synthon [bearing two O-dimethoxytrityl groups] was introduced at the first step of ODN elongation as a short sequence of four branched synthons alternated with three thymidine residues. Conjugates were assessed in a DNA sandwich hybridization test developed for hepatitis B virus detection. Sensitivity limits were evaluated and compared to those obtained with an other polymer, poly(maleic anhydride-alt-methyl vinyl ether) [poly(MA/MVE)]. A sensitivity limit of 2.6 ϫ 10 7 DNA copies/mL was reached with the poly(MA/MVE)-ODN conjugate at the capture phase and with the poly(NAM/NAS)-branched ODN conjugate at the detection phase of the test.

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“…The solvents were then removed in vacuo, and the conjugate was resuspended in 200 L of water. Conjugate [1] was used as described for ELOSA assays.…”

Section: Synthesis Of Poly(ma/mve)-odn Conjugate [1]mentioning

confidence: 99%

Section: Estimation Of Odn Number Per Polymer Chainmentioning

confidence: 99%

Section: Detection Of Hbv Dna By the Elosa Testmentioning

confidence: 99%

Section: Assessment Of the Various Conjugate Performances In An Elosamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Oligonucleotide synthesis onto poly(N‐acryloylmorpholine‐co‐N‐acryloxysuccinimide): Assessment of the resulting conjugates in a DNA sandwich hybridization test

Minard‐Basquin

Chaix

D’Agosto

et al. 2004

J of Applied Polymer Sci

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Designing primers from multiple sequences using matchup program to improve detection of hepatitis B virus by polymerase chain reaction

et al. 2010

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Traditionally primers for PCR detection of viruses have been selected from genomic sequence of single or representative viral strain. However, high mutation rate of viral genomes often results in failure in detecting viruses in clinical and environmental samples. Thus, it seems necessary to consider primers designed from multiple viral sequences in order to improve detection of viral variants. Matchup is a program intended to select universal primers from multiple sequences. We designed using Matchup program primer pairs for HBV detection from 691 full genomic HBV DNA sequences available from NCBI GenBank database. Thousands of primer candidates were initially extracted and these were sequentially filtered down to 5 primer pairs. These primer pairs were tested by PCR using 5 HBV Korean HBsAg(+) patient sera, and eventually one universal primer pair was selected and named MUW (multiple-universal-worldwide). This primer pair, 3 HBV reference primer pairs reported by others and 1 commercial primer pair were compared using 86 HBV HBsAg(+) sera from Korean and Vietnamese patients. The detection rate for MUW primer pair was 72.1%, much greater than those obtained by reference and commercial primers (32.5 to 40.7%). The superiority of MUW primer pair appeared to be correlated with the conserved sequences of the forward primer binding sites and primer quality score. These results suggest that the universal primers designed by the Matchup program from multiple sequences could be useful in detecting viruses from clinical samples.

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Routine detection and quantification of hepatitis B virus DNA in clinical laboratories: performance of three commercial assays

Pawlotsky

Bastie

Hézode

et al. 2000

Journal of Virological Methods

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Quantitative Detection of Hepatitis B Virus DNA with a New PCR Assay

Cited by 6 publications

References 28 publications

Oligonucleotide synthesis onto poly(N‐acryloylmorpholine‐co‐N‐acryloxysuccinimide): Assessment of the resulting conjugates in a DNA sandwich hybridization test

Oligonucleotide synthesis onto poly(N‐acryloylmorpholine‐co‐N‐acryloxysuccinimide): Assessment of the resulting conjugates in a DNA sandwich hybridization test

Designing primers from multiple sequences using matchup program to improve detection of hepatitis B virus by polymerase chain reaction

Routine detection and quantification of hepatitis B virus DNA in clinical laboratories: performance of three commercial assays

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