A new primer set improves the efficiency of competitive PCR–ELISA for the detection of B19 DNA

Bonvicini, Francesca; Gallinella, Giorgio; Cricca, Monica; Venturoli, Simona; Musiani, Monica; Zerbini, Marialuisa

doi:10.1016/j.jcv.2003.09.002

Cited by 4 publications

(2 citation statements)

References 9 publications

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“…6 Recently the detection of DNA by probes that can reconstitute into active peroxidase-mimicking G-quadruplex DNAzymes (Figure 1), 7 has gained increasing popularity because these approaches afford a simple detection of nucleic acids via colorimetric means without the need for expensive proteins such as avidin-horseradish peroxidases conjugates. 8 The use of DNA peroxidases for biomolecule detection in homogeneous formats eliminate nonspecific binding phenomenon associated with protein-based biocatalyst, obviate the need for expensive refrigerated storage, and reduce the number of analytical steps for biomolecule sensing. So far two classes of G-rich probes, symmetric (structure A, Figure 1) 7b,c and asymmetric (structure B, Figure 1), 7d have been used to detect DNA analytes.…”

Section: Introductionmentioning

confidence: 99%

Colorimetric Split G-Quadruplex Probes for Nucleic Acid Sensing: Improving Reconstituted DNAzyme’s Catalytic Efficiency via Probe Remodeling

2009

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Split G-rich DNA probes can assemble into active peroxidase-mimicking DNAzymes in the presence of bioanalytes such as DNA, thereby providing a simple and cheap means to detect analytes in biological samples. A comprehensive study designed to reveal the salient probe architectural features and reaction conditions that facilitate facile reconstitution into enzymatically proficient enzymes unveiled these important findings: (a) The loops that connect the G3-tracts in a G-quadruplex structure can be replaced with a stem-loop or loop-stem-loop motif without destabilizing the resulting quadruplex structure; endowing the split G-rich probes with regions of limited complementarity leads to more proficient reconstituted enzymes. (b) The addition of hemin to antiparallel G-quadruplex DNAzymes lead to a blue shift in the CD spectra of the G-quadruplex DNAzymes. (c) The architectures of the DNA motifs that lie adjacent to the G-quadruplex structure influence both the stability and the enzymatic proficiency of the reconstituted enzymes. (d) The nature of the monovalent cation that is present in excess is a key determinant of the turnover number of the G-quadruplex DNAzyme; decomposition of G-quadruplex DNAzymes is slower in buffers that contain ammonium ions than those that contain sodium or potassium ions. These findings are important for the design of bioassays that use peroxidase-mimicking G-quadruplexes as detection labels.

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Section: Introductionmentioning

confidence: 99%

Colorimetric Split G-Quadruplex Probes for Nucleic Acid Sensing: Improving Reconstituted DNAzyme’s Catalytic Efficiency via Probe Remodeling

2009

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“…Positive and negative archival samples were collected in our laboratory in the year 2004 and tested previously by PCR-ELISA for B19 virus DNA (16 ). The positive archival samples comprised 5 bone marrow aspirates from anemic patients whose blood previously tested positive for B19 DNA by PCR-ELISA, 5 amniotic fluid cell samples from patients with fetal hydrops whose amniotic fluid previously tested positive for B19 DNA by PCR-ELISA, and 3 paraffin-embedded liver biopsy sections from transplant recipients whose biopsy lysates previously tested positive for B19 DNA by PCR-ELISA.…”

Section: Archival and Clinical Samplesmentioning

confidence: 99%

Peptide Nucleic Acid–Based In Situ Hybridization Assay for Detection of Parvovirus B19 Nucleic Acids

Bonvicini¹,

Filippone²,

Manaresi³

et al. 2006

Clinical Chemistry

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Background: Peptide nucleic acid (PNA) molecules are known to bind complementary nucleic acid sequences with a much stronger affinity and with more stable binding than DNA or RNA molecules. We chose parvovirus B19, which is diagnosed by detection of nucleic acids by in situ hybridization assay (ISH) and/or PCR, as an experimental model to develop an ISH assay that uses biotinylated PNA probes to detect viral genome in clinical specimens. Methods: We first optimized the PNA-ISH assay on B19-infected and mock-infected UT-7/EpoS1 cells and then tested the assay on archival B19 specimens and on consecutive specimens. All data were compared with data obtained with a standardized DNA-based ISH assay and confirmed by a PCR-ELISA. Results: PNA-ISH detected B19 genome in a higher number of B19-infected UT-7/EpoS1 cells and with a more defined localization of viral nucleic acids than the standardized DNA-ISH assay. Moreover, PNA-ISH was able to detect B19 genome in all positive archival samples, whereas DNA-ISH failed in 5 samples. PNA-ISH detected more positive samples than DNA-ISH when consecutive specimens were analyzed, and a close agreement was found with PCR-ELISA results. Conclusions: The PNA-ISH assay had sensitivity and specificity comparable to a PCR assay and was more practical and quicker to perform than standard hybridization assays. The assay may be a suitable diagnostic

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PCR–ELISA for High-Throughput Blood Group Genotyping

St‐Louis

2009

DNA and RNA Profiling in Human Blood

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During the last decade, blood bank specialists have shown an increased interest in molecular analyses to complement serology work in determining blood group antigens. To efficiently respond to the numerous demands made for hemolytic disease of the newborn cases and polytransfused patients, we designed an inexpensive colorimetric high-throughput method to genotype several blood group antigens rapidly. Three simple steps are required to perform this technique: genomic DNA extraction, PCR amplification, and amplicon detection by a microplate ELISA. The 96-well plate format facilitates the manipulations and enables the analysis of multiple samples at once or the analysis of multiple antigens for fewer samples.The most common and clinically relevant minor blood group antigens were adapted to this method and are described in this work: Rh (D, C, c, E, e), Kell (K, k), Duffy (Fy(a), Fy(b)), and Kidd (Jk(a), Jk(b)). Other blood group antigens could be easily tested this way as long as their molecular basis is well established.

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A new primer set improves the efficiency of competitive PCR–ELISA for the detection of B19 DNA

Cited by 4 publications

References 9 publications

Colorimetric Split G-Quadruplex Probes for Nucleic Acid Sensing: Improving Reconstituted DNAzyme’s Catalytic Efficiency via Probe Remodeling

Colorimetric Split G-Quadruplex Probes for Nucleic Acid Sensing: Improving Reconstituted DNAzyme’s Catalytic Efficiency via Probe Remodeling

Peptide Nucleic Acid–Based In Situ Hybridization Assay for Detection of Parvovirus B19 Nucleic Acids

PCR–ELISA for High-Throughput Blood Group Genotyping

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