Rapid and specific DNA detection by magnetic field-enhanced agglutination assay

Diagnostic Performance of a Magnetic Field-Enhanced Agglutination Readout in Detecting Either Viral Genomes or Host Antibodies in Arbovirus Infection

“…The MAMD forward primer (5 AAC ATG GGR AAR AGR GAR AA3 ) was 5 -tagged with biotin to generate biotinylated amplified genomes. Asymmetric RT-PCR amplification was carried out using 5 µL of extracted viral RNAs mixed with 3 µL of MAMD forward primer (10 µM) and 0.3 µL of cFD2 reverse primer (5 GTG TCC CAG CCG GCG GTG TCA GC3 ; 10 µM) in a final volume of 50 µL [12,14]. The total amplification time was 2.5 h [12].…”

“…Asymmetric RT-PCR amplification was carried out using 5 µL of extracted viral RNAs mixed with 3 µL of MAMD forward primer (10 µM) and 0.3 µL of cFD2 reverse primer (5 GTG TCC CAG CCG GCG GTG TCA GC3 ; 10 µM) in a final volume of 50 µL [12,14]. The total amplification time was 2.5 h [12]. The PCR procedures were performed using a T Advanced Biometra thermal cycler (Analytik Jena AG, Germany) and amplified DENV DNAs were tested immediately or stored at −20 • C until their use.…”

“…The 5'-tetrathiolated DENV probe 5 TCC TTC YAC TCC RCT3 was synthesized on a 1 µmol scale using a DNA synthesizer, and lyophilized before use [11,12]. This DENV probe, designed to detect the four serotypes of DENV genomes, was covalently grafted onto 200 nm diameter MNPs (200 nm carboxyl-adembeads, Ademtech, Pessac France) using an amino-polyethylene glycol (PEG)-maleimide crosslinker as previously described [12]. The MNPs were passivated by incubations with 1 mL of 1.5 M Tris-HCl, pH 8.8, for 20 min and 250 µL of a cysteine solution (80 mg/mL) for 10 min.…”

“…The molecular detection step was performed in a disposable spectrophotometric cuvette surrounded by an electromagnet that provided a 15 mT magnetic field. This prototype included a LED source emitting at 650 nm and a photodiode to perform a simple optical detection [12].…”

“…In order to develop a fast and low-cost DNA detection approach avoiding fluorescence readout, we more recently carried out a proof-of-concept study using synthetic DNA sequences or cultivated viruses to describe a simple detection readout by magnetic fieldenhanced agglutination (MFEA) [12].…”