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Sequence specific visual detection of LAMP reactions by addition of cationic polymers.

ABSTRACT: BACKGROUND: Development of a practical gene point-of-care testing device (g-POCT device) requires innovative detection methods for demonstrating the results of the gene amplification reaction without the use of expensive equipment. We have studied a new method for the sequence-specific visual detection of minute amounts of nucleic acids using precipitation reaction by addition of cationic polymers to amplicons of Loop mediated isothermal Amplification (LAMP). RESULTS: Oligo DNA probes labeled with different fluorescent dyes were prepared for multiple nucleic acid templates, and the templates were amplified by the LAMP reactions under the existence of the probes. At completion of the LAMP reaction, an optimal amount of low molecular weight polyethylenimine (PEI) was added, resulting in the precipitation of the insoluble LAMP amplicon-PEI complex. The fluorescently labeled Oligo DNA probes hybridized to the LAMP product were incorporated into the precipitation, and the precipitate emitted fluorescence corresponding to the amplified nucleic acid templates. The color of emitted fluorescence can be detected easily by naked eye on a conventional UV illuminator. CONCLUSION: The presence or absence of minute amount of nucleic acid templates could be detected in a simple manner through visual assessment for the color of the LAMP amplicon-PEI complex precipitate. We conclude that this detection method may facilitate development of small and simple g-POCT device.

SUBMITTER: Mori Y

PROVIDER: S-EPMC1373654 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Sequence specific visual detection of LAMP reactions by addition of cationic polymers.

Mori Yasuyoshi Y Hirano Tsuyoshi T Notomi Tsugunori T

BMC biotechnology 20060110

<h4>Background</h4>Development of a practical gene point-of-care testing device (g-POCT device) requires innovative detection methods for demonstrating the results of the gene amplification reaction without the use of expensive equipment. We have studied a new method for the sequence-specific visual detection of minute amounts of nucleic acids using precipitation reaction by addition of cationic polymers to amplicons of Loop mediated isothermal Amplification (LAMP).<h4>Results</h4>Oligo DNA prob ...[more]

PMID: 16401354

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Project description:The radical polymerization of acid-washed and unwashed softwood kraft lignin with [2-(methacryloyloxy) ethyl] trimethylammonium chloride (METAC) was attempted to investigate the production of lignin-based flocculants for simulated wastewater. The incorporation of METAC onto lignin resulted in a cationic charge density (2.3-3.3 meq/g), increased water solubility (89-96% in neutral pH), and increased molecular weight (70,000-210,000 g/mol) of lignin. The lignin-METAC polymers generated from acid-washed lignin had higher molecular weights than those generated from unwashed lignin. The lignin-METAC polymers showed lower resistance to thermal decomposition than unmodified lignin due to the inclusion of PolyMETAC. The unmodified acid-washed lignin samples did not significantly affect the COD of the wastewater, while the unmodified unwashed lignin samples contributed to the COD, implying that unmodified lignin was not suitable for wastewater treatment. The flocculation of wastewater with lignin-METAC led to the chemical oxygen demand (COD) reduction of 17-23% and total organic carbon (TOC) drop of 51-60%. The lignin-METAC polymer with the highest molecular weight (produced from acid-washed lignin) reached the highest COD removal, while lignin-METAC polymer with the highest charge density (produced from unwashed lignin) reached the highest TOC removal. Focused beam reflectance measurement (FBRM) studies revealed that the lignin-METAC polymer produced from acid-washed lignin with a high molecular weight generated larger and more flocs in wastewater than the lignin-METAC polymer produced from unwashed lignin. The comparison of theoretical and experimental dosages required for neutralizing the charges of wastewater demonstrated that charge neutralization was the main flocculation mechanism, although a bridging mechanism was also involved for component removals from wastewater. The use of 1 mg/L of alum along with 65 mg/L lignin-METAC in a dual coagulation-flocculation system led to higher average phosphorous (42%) and COD (44%) removals than the singular flocculation system only using 65 mg/L of lignin-METAC (with phosphorous removals of 3.4% and COD removals of 18.7%). However, lignin-METAC flocculant slightly increased the ammonia-nitrogen content in both singular flocculation and dual coagulation-flocculation systems due to the residual ammonia content of lignin-METAC. The coagulation-flocculation system determined that the use of lignin-METAC (65 mg/L) could reduce the alum dosage significantly while maintaining a similar organic content reduction of 44% for wastewater.

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Sequence specific visual detection of LAMP reactions by addition of cationic polymers.

Publications

Sequence specific visual detection of LAMP reactions by addition of cationic polymers.

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