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A single-tube nucleic acid extraction, amplification, and detection method using aluminum oxide.

ABSTRACT: A disposable 0.2-ml polymerase chain reaction (PCR) tube modified with an aluminum oxide membrane (AOM) has been developed for the extraction, amplification, and detection of nucleic acids. To assess the dynamic range of AOM tubes for real-time PCR, quantified herpes simplex virus (HSV) DNA was used to compare AOM tubes to standard PCR tubes. AOM PCR tubes used for amplification and detection of quantified HSV-1 displayed a crossing threshold (C(T)) shift 0.1 cycles greater than PCR tube controls. Experiments with HSV-1-positive cerebrospinal fluid (CSF) examined the extraction, amplification, and detection properties of the AOM tubes compared to the Qiagen DNA blood mini kit. The AOM extraction, amplification, and detection of HSV-1 in CSF displayed differences of less than one C(T) when compared to Qiagen-extracted samples. Experiments testing the AOM method using clinical CSF samples displayed 100% concordance with reported results. AOM tubes have no adverse effects on amplification or fluorescence acquisition by real-time PCR and can be effectively used for the extraction, amplification, and detection of HSV from CSF. The AOM single tube method is a fast, reliable, and reproducible technique for the extraction, amplification, and detection of HSV in CSF.

SUBMITTER: Dames S

PROVIDER: S-EPMC1867575 | biostudies-literature | 2006 Feb

REPOSITORIES: biostudies-literature

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A single-tube nucleic acid extraction, amplification, and detection method using aluminum oxide.

Dames Shale S Bromley L Kathryn LK Herrmann Mark M Elgort Marc M Erali Maria M Smith Roger R Voelkerding Karl V KV

The Journal of molecular diagnostics : JMD 20060201 1

A disposable 0.2-ml polymerase chain reaction (PCR) tube modified with an aluminum oxide membrane (AOM) has been developed for the extraction, amplification, and detection of nucleic acids. To assess the dynamic range of AOM tubes for real-time PCR, quantified herpes simplex virus (HSV) DNA was used to compare AOM tubes to standard PCR tubes. AOM PCR tubes used for amplification and detection of quantified HSV-1 displayed a crossing threshold (C(T)) shift 0.1 cycles greater than PCR tube control ...[more]

PMID: 16436630

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Project description:BACKGROUND: Isothermal amplification methods provide alternatives to PCR that may be preferable for some nucleic acid target detection tasks. Among current isothermal target detection methods, ramified rolling circle amplification (RAM) of single-stranded DNA circles that are formed by ligation of linear DNA probes (C-probes or padlock probes) offers a unique target detection system by linked primers and a simple amplification system that is unconstrained by the target's sequence context. Earlier implementations of RAM-based target detection were reported to be limited by background noise, due in part to unligated C-probe in the amplification reaction. We show here that a target-detection system using a biotinylated target-capture probe together with automated bead-handling reduces or eliminates background amplification noise. We demonstrate the system's performance by detection of a single-nucleotide polymorphism in human genomic DNA. METHODOLOGY: Target detection by RAM entails hybridization and ligation of a C-probe, followed by amplification and RAM signal detection. We evaluated RAM target detection in genomic DNA using recognition of a human Factor V gene single nucleotide polymorphism (G1691A) as a model. Locus-specific C-probes were annealed and ligated to genomic DNAs that represent the 3 possible genotypes at this locus, then ligated C-probes were amplified by real time RAM. The majority of the steps in the assay were performed with a magnetic bead-based chemistry on an automated platform. We show that the specificity of C-probe ligation permits accurate genotyping of this polymorphism. The assay as described here eliminates some of the background noise previously described for C-probe ligation, RAM amplification assays. CONCLUSION: The methods and results presented here show that a combination of C-probe detection, automated sample processing, and isothermal RAM amplification provide a practical approach for detecting DNA targets in complex mixtures.

Project description:BACKGROUND: The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. PRINCIPAL FINDINGS: Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. CONCLUSIONS: The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings.

Dataset Information

A single-tube nucleic acid extraction, amplification, and detection method using aluminum oxide.

Publications

A single-tube nucleic acid extraction, amplification, and detection method using aluminum oxide.

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