Multiplex primer prediction software for divergent targets.

We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets to amplify all members of large, diverse and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers ( approximately 3700 18-mers or approximately 2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus (FMDV), hemagglutinin (HA) and neuraminidase (NA) segments of influenza A virus, Norwalk virus, and HIV-1. We empirically demonstrated the application of the software with a multiplex set of 16 short (10 nt) primers designed to amplify the Poxviridae family to produce a specific amplicon from vaccinia virus.

In vitro double transposition for DNA identification.

We present a double transposition technique that inserts two different transposons into target DNA to act as priming sites for amplifying the region between the two transposons for sequencing applications. Unlike some current sequencing approaches, the genome of the unknown target remains intact in this method. The transposition reaction, DNA repair, and subsequent sequencing were performed entirely in vitro, without the need for transformation into bacteria, and resulted in sequence homology with the plasmid DNA target. This approach can reduce the time required for the assay by more than a day compared with standard techniques and reduces the number of required enzymatic steps. In addition, the in vitro method enables transposition to be carried out in automated microfluidic platforms without the need for significant sample manipulation. As a demonstration of incorporating transposition techniques into high-throughput technologies, single transposition reactions were carried out in picoliter-sized droplets generated on a microfluidic platform.

High level of HIV false positives using EIA-based algorithm in survey: Importance of confirmatory testing.

The Mozambique Indicators of Immunization, Malaria and HIV/AIDS (IMASIDA) survey was conducted in 2015 and used a two Enzyme Immunoassay (EIA) (Vironostika HIV-1/2 and Murex HIV-1/2) based algorithm to determine the HIV status of the consented participants. The Mozambique Ministry of Health, with support from the US Centers for Disease Control and Prevention (US CDC), added Bio-Rad Geenius™ HIV-1/2 Supplemental Assay to the IMASIDA HIV testing algorithm to confirm all specimens that were found to be reactive on one or both EIAs. In total 11690 specimens were collected to estimate the proportion of HIV positive samples. Results indicate that the proportion of HIV positive samples based on the concordant positive results of two EIA assays was 21.5% (2518/11690). The addition of the Geenius assay to the IMASIDA HIV testing algorithm demonstrated that 792 (31.5%) of 2518 specimens were false-positive and reduced the proportion of HIV positive samples to 14.7% (1722/11690), demonstrating the importance of including a highly specific HIV test to confirm HIV diagnosis. HIV surveys exclusively based on EIA testing algorithm may result in misleading high prevalence results. Our results demonstrate that more specific confirmatory testing should be added to the EIA-based algorithms to ensure accurate HIV diagnosis and correct HIV prevalence estimate in cross-sectional surveys.

Magnetic bead based immunoassay for autonomous detection of toxins.

We are developing an automated system for the simultaneous, rapid detection of a group of select agents and toxins in the environment. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag through a cleavable linkage. Aqueous samples are incubated with the mixture of antibodies along with streptavidin-coated magnetic beads and a photoactive porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactive group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags. Released eTags are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated in a flow-through format with higher LODs of 32 ng/mL (or 640 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.

Microdissection with PCR in situ.

In situ amplification techniques are designed to increase the mass of DNA in a fixed target, either whole cells or tissue sections. When combined with fluorescently labeled nucleotides, they can be used for locus detection. They also can be used to increase target mass for subsequent operations, such as cellular or chromosomal isolation by microdissection. When combined with chromosome microdissection, these techniques allow libraries to be made from single copies of chromosomes, chromosome fragments, or even bacteria.

Effects of seasonal allergic rhinitis on fatigue levels and mood.

OBJECTIVE: Many allergy patients complain of fatigue, moodiness, and dysphoria during their allergy seasons. This study evaluated the effect of symptomatic allergic rhinitis on both fatigue level and mood. METHOD: Symptomatic ragweed allergic rhinitis patients on no medications and healthy control subjects completed the Multi-Dimensional Fatigue Inventory and the Positive Affect-Negative Affect mood rating scales in an in-out-in ragweed season research design. RESULTS: During ragweed seasons, allergic patients reported higher levels of general fatigue and mental fatigue, but not physical fatigue, as well as reduced motivation. Patients described experiencing feelings of greater sadness and reduced pleasurable engagement. Increased anxiety or emotional distress was not reported. CONCLUSIONS: These findings suggest that having allergic reactions to ragweed pollen causes significant fatigue and mood changes in at least a subgroup of patients. Psychoneuroimmunology and medical genetics research suggests that allergic reactions engender biochemical changes that directly affect the central nervous system.