Covalent immobilization of DNA onto polystyrene microwells: The molecules are only bound at the 5′ end

Rasmussen, Steen; Larsen, Mette Ravn; Rasmussen, Svend Erik

doi:10.1016/0003-2697(91)90518-x

Cited by 168 publications

(78 citation statements)

References 14 publications

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“…A disadvantage is that capture probes are bound more or less efficiently to the microwell at multiples sites. The ideal immobilization of the probe onto the polystyrene surface is obtained by covalent binding at only one point preferably located at either the 3Ј or the 5Ј end (17).…”

Section: Discussionmentioning

confidence: 99%

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Development of a PCR Assay Followed by Nonradioactive Hybridization Using Oligonucleotides Covalently Bound to CovaLink NH Microwells for Detection of Four Plasmodium Species in Blood Samples from Humans

et al. 2006

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We developed and evaluated a PCR-based assay to detect four Plasmodium species in 79 blood samples from 56 travelers returning from areas where malaria is endemic. DNA amplification targeting a small region of the 18S rRNA gene was performed with Plasmodium genus-specific primers. The biotinylated PCR products were then identified by PCR-colorimetric Covalink NH microwell plate hybridization (CMPH) using species-specific phosphorylated probes covalently bound to a pretreated polystyrene surface. The results from PCR-CMPH showed high specificity, and for 47 of the 56 patients (84%), microscopy and PCR-CMPH results were in agreement. Discordant results were reevaluated with microscopy examination, other molecular methods, and DNA sequencing. Except for one patient, discrepancies were resolved in favor of PCR-CMPH: three mixed infections were detected, four species identification errors were corrected, and two negative results were shown to be positive. Our results indicate that PCR-CMPH is a simple, rapid, and specific method for malaria diagnosis. It employs stable reagents and inexpensive equipment, making it suitable for routine epidemiological use.

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Section: Discussionmentioning

confidence: 99%

“…scribed previously by Rasmussen et al (17). Briefly, each phosphorylated probe (concentrations between 25 and 75 pmol/well) was denatured for 10 min at 95°C and rapidly cooled on ice to prevent secondary structure formation.…”

mentioning

confidence: 99%

Development of a PCR Assay Followed by Nonradioactive Hybridization Using Oligonucleotides Covalently Bound to CovaLink NH Microwells for Detection of Four Plasmodium Species in Blood Samples from Humans

et al. 2006

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“…When the DNA concentration is low, dryadsorption resulted in the binding of a DNA molecule to the microwell surface at multiple points, whereas the ideal immobilization of a DNA molecule is binding at only one point. 24,25 In the second method, an immobilization buffer containing 1.5 M NaCl is used. 1 A high ion strength is employed so as to decrease the electric repulsion between polyanionic DNA.…”

Section: Selection Of Immobilization Methodsmentioning

confidence: 99%

Time-Resolved Fluorometric Detection of DNA Using a Tetradentate β-Diketonate Europium Chelate as a Label

et al. 1999

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“…The capture probe was produced by PCR using: (1) the PVYF-PVYR4 amplicon obtained from strain PVY O (PVY O-0343 , Table 1) and cloned in plasmid vector (TA cloning kit, Invitrogen) as the target; (2) a forward primer internal to the PVYF-PVYR4 amplicon and (3) the PVYR4 primer. The capture probe was denatured and immobilized on the plastic of the plate (Covalink, Nunc) using the protocol described previously (Rasmussen et al, 1991). The single strand detection probe (5% GAATGCAAAACCAACACTTAGGCAAAT-CATGGCACA 3%) biotinylated at its 3% end was selected in a region of the PVYF-PVYR4 amplicon non complementary to the capture probe and used at 25 ng per well.…”

Section: Post-rt-pcr Detectionmentioning

confidence: 99%

RT-PCR-ELOSA tests on pooled sample units for the detection of virus Y in potato tubers

Chandelier

Dubois

Baelen

et al. 2001

Journal of Virological Methods

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A highly sensitive RT-PCR protocol able to detect potato virus Y (PVY) in pooled sample units (tubers) was developed. PVY-specific primers selected in the coat protein gene were found to amplify a 359 bp fragment from diluted crude extract of infected tubers. For the detection of the amplification products, a colorimetric detection procedure in microtiter plates was established. The amplicons are hybridized between a covalently linked capture probe and a specific biotinylated detection probe ELOSA tests. This detection method detects at least 50 pg of virus per reaction for the four cultivars tested. The RT-PCR-ELOSA assay was adapted to pooled units in order to increase the sample size while reducing the number of tests.

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Covalent immobilization of DNA onto polystyrene microwells: The molecules are only bound at the 5′ end

Cited by 168 publications

References 14 publications

Development of a PCR Assay Followed by Nonradioactive Hybridization Using Oligonucleotides Covalently Bound to CovaLink NH Microwells for Detection of Four Plasmodium Species in Blood Samples from Humans

Development of a PCR Assay Followed by Nonradioactive Hybridization Using Oligonucleotides Covalently Bound to CovaLink NH Microwells for Detection of Four Plasmodium Species in Blood Samples from Humans

Time-Resolved Fluorometric Detection of DNA Using a Tetradentate β-Diketonate Europium Chelate as a Label

RT-PCR-ELOSA tests on pooled sample units for the detection of virus Y in potato tubers

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