A rapid, instrument-free, sample-to-result nucleic acid amplification test.

The prototype demonstrated here is the first fully integrated sample-to-result diagnostic platform for performing nucleic acid amplification tests that requires no permanent instrument or manual sample processing. The multiplexable autonomous disposable nucleic acid amplification test (MAD NAAT) is based on two-dimensional paper networks, which enable sensitive chemical detection normally reserved for laboratories to be carried out anywhere by untrained users. All reagents are stored dry in the disposable test device and are rehydrated by stored buffer. The paper network is physically multiplexed to allow independent isothermal amplification of multiple targets; each amplification reaction is also chemically multiplexed with an internal amplification control. The total test time is less than one hour. The MAD NAAT prototype was used to characterize a set of human nasal swab specimens pre-screened for methicillin-resistant Staphylococcus aureus (MRSA) bacteria. With qPCR as the quantitative reference method, the lowest input copy number in the range where the MAD NAAT prototype consistently detected MRSA in these specimens was ∼5 × 10(3) genomic copies (∼600 genomic copies per biplexed amplification reaction).

Sensitive detection assays for influenza RNA do not reveal viremia in US blood donors.

BACKGROUND: There have been anecdotal reports of influenza viremia since the 1960s. We present an assessment of the prevalence of seasonal and 2009 H1N1 influenza viremia (via RNA testing) in blood donor populations using multiple sensitive detection assays. METHODS: Several influenza RNA amplification assays, including transcription-mediated amplification (TMA) and 2 reverse-transcription polymerase chain reaction (RT-PCR) assays, were evaluated and used to test donor samples. Retrospective samples from 478 subjects drawn at sites with high influenza activity were tested. Prospective samples were collected from 1004 blood donors who called their donation center within 3 days of donation complaining of influenza-like illness (ILI). The plasma collected on the day of donation for these subjects was tested. RESULTS: Of the repository samples, 2 of 478 plasma samples were initially reactive but not repeat reactive by influenza TMA. Of blood donors reporting ILI symptoms postdonation, 1 of 1004 samples was TMA initially reactive but not repeat reactive; all samples were nonreactive by RT-PCR testing. CONCLUSIONS: Targeting blood donor populations most likely to have influenza infection, we failed to detect influenza RNA in 1482 donor samples, with most tested by 3 different RNA assays. Seasonal influenza does not appear to pose a significant contamination threat to the blood supply.

Improved biplex quantitative real-time polymerase chain reaction with modified primers for gene expression analysis.

Stabilizing modified bases incorporated in primers allows the reduction of housekeeping gene primer concentration not possible with regular primers without sacrificing amplification efficiency. Low primer concentration allows coamplification of the most abundant housekeeping genes with very rare templates without mutual inhibition. Real-time polymerase chain reaction (PCR) coamplification of 18S ribosomal RNA with several genes of interest was used in this study with MGB Eclipse (Nanogen, San Diego, CA) hybridization probes. The results may be useful for high throughput gene expression studies as they simplify validation experiments.

A novel endonuclease IV post-PCR genotyping system.

Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5' end and fluorophore attached to the 3' end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3' end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.

Single nucleotide polymorphism genotyping by two colour melting curve analysis using the MGB Eclipse Probe System in challenging sequence environment.

Probe and primer design for single nucleotide polymorphism (SNP) detection can be very challenging for A-T DNA-rich targets, requiring long sequences with lower specificity and stability, while G-C-rich DNA targets present limited design options to lower GC-content sequences only. We have developed the MGB Eclipse Probe System, which is composed of the following elements: MGB Eclipse probes and primers, specially developed software for the design of probes and primers, a unique set of modified bases and a Microsoft Excel macro for automated genotyping, which ably solves, in large part, this challenge. Fluorogenic MGB Eclipse probes are modified oligonucleotides containing covalently attached duplex-stabilising dihydrocyclopyrroloindole tripeptide (DPI3), the MGB ligand (MGB is a trademark of Epoch Biosciences, Bothell, WA), which has the combined properties of allowing the use of short sequences and providing great mismatch discrimination. The MGB moiety prevents probe degradation during polymerase chain reaction (PCR), allowing the researcher to use real time data; alternatively, hybridisation can be accurately measured by a post-PCR two-colour melt curve analysis. Using MGB Eclipse probes and primers containing modified bases further enhances the analysis of difficult SNP targets. G- or C-rich sequences can be refractory to analysis due to Hoogsteen base pairing. Substitution of normal G with Epoch's modified G prevents Hoogsteen base pairing, allowing both superior PCR and probe-based analysis of GC-rich targets. The use of modified A and T bases allows better stabilisation by significantly increasing the Tm of the oligonucleotides. Modified A creates A-T base pairs that have a stability slightly lower than a G-C base pair, and modified T creates T-A base pairs that have a stability about 30 per cent higher than the unmodified base pair. Together, the modified bases permit the use of short probes, providing good mismatch discrimination and primers that allow PCR of refractory targets. The combination of MGB Eclipse probes and primers enriched with the MGB ligand and modified bases has allowed the analysis of refractory SNPs, where other methods have failed.

Reduced aggregation and improved specificity of G-rich oligodeoxyribonucleotides containing pyrazolo[3,4-d]pyrimidine guanine bases.

Guanine (G)-rich oligodeoxyribonucleotides (ODNs) can form undesired complexes by self association through non-Watson-Crick interactions. These aggregates can compromise performance of DNA probes and make genetic analysis unpredictable. We found that the 8-aza-7-deazaguanine (PPG), a pyrazolo[3,4-d]pyrimidine analog, reduces guanine self association of G-rich ODNs. In the PPG heterocycle, the N-7 and C-8 atoms of G are interposed. This leaves the ring system with an electron density similar to G, but prevents Hoogsteen-bonding associated with N-7. ODNs containing multiple PPG bases were easily prepared using a dimethylformamidine-protected phosphoramidite reagent. Substitution of PPG for G in ODNs allowed formation of more stable DNA duplexes. When one or more PPGs were substituted for G in ODNs containing four or more consecutive Gs, G aggregation was eliminated. Substitution of PPG for G also improved discrimination of G/A, G/G and G/T mismatches in Watson-Crick hybrids. Use of PPG in fluorogenic minor groove binder probes was also explored. PPG prevented aggregation in MGB probes (MGB(TM) is a trademark of Epoch Biosciences) and allowed use of G-rich sequences. An increased signal was observed in 5'-PPG probes due to reduced quenching of fluorescein by PPG. In summary, substitution of PPG for G enhances affinity, specificity, sensitivity and predictability of G-rich DNA probes.

Synthesis of bis-spermine dimers that are potent polyamine transport inhibitors.

A series of novel spermine dimer analogues was synthesized and assessed for their ability to inhibit spermidine transport into MDA-MB-231 breast carcinoma cells. Two spermine molecules were tethered via their N(1) primary amines with naphthalenedisulfonic acid, adamantanedicarboxylic acid and a series of aliphatic dicarboxylic acids. The linked spermine analogues were potent polyamine transport inhibitors and inhibited cell growth cytostatically in combination with a polyamine synthesis inhibitor. Variation in the linker length did not alter polyamine transport inhibition. The amount of charge on the molecule may influence the molecular interaction with the transporter since the most potent spermidine transport inhibitors contained 5-6 positive charges.