Assay For Ultrasensitive Detection Research Articles

Multiplex qPCR assay for ultra sensitive detection of JCV DNA with simultaneous identification of genotypes that discriminates non-virulent from virulent variants

BackgroundJC virus (JCV) is the etiologic agent for progressive multifocal leukoencephalopathy (PML), a demyelinating disease occurring in the brain of patients with underlying immune compromised states. All viable JCV genomes contain a conserved region in the T protein coding nucleotide sequence that when detected by PCR in CSF is a confirmatory diagnostic marker for PML along with clinical and neuroradiological evidence. The non-coding regulatory region (NCRR) is hypervariable, as evidenced by nucleotide sequence of the non-virulent variant, which is predominantly excreted in urine, versus that of virulent variants found in brain and CSF of PML patients. All variants can be found in blood. ObjectiveA single assay that quantifies and identifies JCV DNA in clinical samples and discriminates between variants has significant value to physicians and patients at risk for PML. Study designSeparate primer pairs were tested together to quantitatively detect conserved viral DNA nucleotide sequence in patient samples, while simultaneously detecting the NCRR specific for the non-virulent variant. ResultsIn testing using control plasmids and patients’ CSF, blood, and urine, PML patients predictably demonstrated the non-virulent, archetype NCRR in urine, but virulent NCRR variants in CSF and blood. ConclusionThe JCV qPCR multiplex assay targets two regions in JCV genomes to simultaneously identify and measure viral DNA, as well as distinguish between variants associated with PML and those that are not. The multiplex results could signal risk for PML if patients are viremic with JCV variants closely associated with PML pathogenesis.

Development of a highly sensitive gold nanoparticle probe-based assay for bluetongue virus detection

A simple gold nanoparticle (GNP) probe based assay (GNPA) that was modified from a bio-barcode assay (BCA) technique, was developed for ultra-sensitive, rapid detection of the bluetongue virus (BTV) VP7 outer-core protein. This assay captures the VP7 target antigen using the GNP probe coated with anti-VP7 polyclonal antibodies and single-stranded signal DNA. Magnetic microparticle (MMP) probes coated with anti-VP7 monoclonal antibodies were then added to form a sandwich immuno-complex. The single-stranded signal DNA coated onto the GNP probe present in the immuno-complex could then be detected by PCR and real-time fluorescence PCR using a TaqMan probe. The assay has a purified VP7 detection limit of 10(-2)fg/ml which is 8 orders of magnitude greater than that of conventional antigen capture ELISAs and 1 order of magnitude more sensitive than that of a conventional BCA. These results indicate that the GNPA is a highly sensitive method for easy detection of BTV proteins and that it can be modified as needed to measure the presence of other proteins.