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Highly rapid amplification-free and quantitative DNA imaging assay.

ABSTRACT: There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a 'sample-in-result-out' mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5?kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20??L. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5?pM and a linear detection range spanning three orders of magnitude.

SUBMITTER: Klamp T

PROVIDER: S-EPMC3655336 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Highly rapid amplification-free and quantitative DNA imaging assay.

Klamp Tobias T Camps Marta M Nieto Benjamin B Guasch Francesc F Ranasinghe Rohan T RT Wiedemann Jens J Petrášek Zdeněk Z Schwille Petra P Klenerman David D Sauer Markus M

Scientific reports 20130101

There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a 'sample-in-result-out' mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in ...[more]

PMID: 23677392

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Project description:A disease known as covert mortality disease has become an increasing problem in the shrimp farming industry in recent years in China and several countries of Southeast Asia, leading to serious losses in production. Litopenaeus vannamei (also known as Pacific white shrimp) is affected by this disease that leads to a range of clinical symptoms including hepatopancreas atrophy and necrosis, soft shell, slow growth, and abdominal muscle whitening and necrosis in the acute stage of disease. A new nodavirus, termed covert mortality nodavirus (CMNV), has been shown to be the etiological agent. In this study, we report a sensitive and specific real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and quantitative detection of CMNV. The optimal conditions for this newly developed RT-LAMP reaction were found to be 6mM MgCl2 and 1.6mM dNTPs, an incubation temperature of 65°C and a reaction time of 50min. The analytical sensitivity of the RT-LAMP assay was estimated to be 6.3pg total RNA of CMNV-infected shrimp and 27 copies of the target plasmid. The diagnostic sensitivity and specificity of the newly developed assay versus the standard nested reverse transcription PCR (RT-PCR) assay was 96.4% and 94.4%, respectively. The reaction products were detected by visual inspection after staining with an in-tube DNA fluorescent dye, a measure taken to eliminate the risk of contamination. The quantitative RT-LAMP assay for CMNV showed high correlation coefficient (r2=0.9953) when the initial templates were above 1000 copies, however the correlation coefficient decreased when the initial templates were lower than 1000 copies. Test of viral load in shrimp indicated that the viral loads varied from 1.5×102 to 6.7×106 copies per mg of cephalothorax tissue. Thus, the CMNV RT-LAMP assay is a sensitive and specific new tool for the field detection and quantification of CMNV in the diagnosis and surveillance of covert mortality disease.

Dataset Information

Highly rapid amplification-free and quantitative DNA imaging assay.

Publications

Highly rapid amplification-free and quantitative DNA imaging assay.

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