Method for lysis and paper-based elution-free DNA extraction with colourimetric isothermal amplification.

Lee, Soo Min; Doeven, Egan H; Yuan, Dan; Guijt, Rosanne M

Lee, Soo Min; Doeven, Egan H; Yuan, Dan; Guijt, Rosanne M.

Afiliación

Lee SM; Centre for Regional and Rural Futures (CeRRF), Deakin University, Locked Bag 20000, Geelong, VIC, 3220, Australia.
Doeven EH; Centre for Regional and Rural Futures (CeRRF), Deakin University, Locked Bag 20000, Geelong, VIC, 3220, Australia.
Yuan D; School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, Waurn Ponds, VIC, 3216, Australia.
Guijt RM; Centre for Regional and Rural Futures (CeRRF), Deakin University, Locked Bag 20000, Geelong, VIC, 3220, Australia. d.yuan@uq.edu.au.

Sci Rep ; 14(1): 14479, 2024 06 24.

Article en En | MEDLINE | ID: mdl-38914553

ABSTRACT

ABSTRACT

Nucleic acid amplification testing has great potential for point-of-need diagnostic testing with high detection sensitivity and specificity. Current sample preparation is limited by a tedious workflow requiring multiple steps, reagents and instrumentation, hampering nucleic acid testing at point of need. In this study, we present the use of mixed cellulose ester (MCE) paper for DNA binding by ionic interaction under molecular crowding conditions and fluid transport by wicking. The poly(ethylene) glycol-based (PEG) reagent simultaneously provides the high pH for alkaline lysis and crowding effects for ionic binding of the DNA under high salt conditions. In this study, we introduce Paper-based Abridged Solid-Phase Extraction with Alkaline Poly(ethylene) Glycol Lysis (PASAP). The anionic mixed cellulose ester (MCE) paper is used as solid phase and allows for fluid transport by wicking, eliminating the need for pipetting skills and the use of a magnet to retain beads. Following the release of DNA from the cells due to the lytic activity of the PASAP solution, the DNA binds to the anionic surface of the MCE paper, concentrating at the bottom while the sample matrix is transported towards the top by wicking. The paper was washed by dipping it in 40% isopropanol for 10 s. After air-drying for 30 s, the bottom section of the paper (3 mm × 4 mm) was snapped off using the cap of a PCR tube and immersed in the colourimetric loop-mediated isothermal amplification (cLAMP) solution for direct amplification and colourimetric detection. The total sample processing was completed in 15 min and ready for amplification. cLAMP enabled the detection of 102 CFU/mL of Escherichia coli (E. coli) from culture media and the detection of E. coli in milk < 103 CFU/mL (10 CFU) after incubation at 68 °C for 60 min, demonstrating applicability of the method to complex biological samples.

Asunto(s)

Técnicas de Amplificación de Ácido Nucleico; Papel; Técnicas de Amplificación de Ácido Nucleico/métodos; Colorimetría/métodos; ADN; Extracción en Fase Sólida/métodos; Polietilenglicoles/química; ADN Bacteriano/aislamiento & purificación; ADN Bacteriano/genética; Técnicas de Diagnóstico Molecular

Palabras clave

E. coli detection; Colourimetric loop-mediated isothermal amplification (cLAMP); Elution-free sample preparation; Milk; Nucleic acid amplification test (NAAT); PASAP; PONT; Paper-based DNA extraction; Point-of-need testing

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Papel / Técnicas de Amplificación de Ácido Nucleico Idioma: En Revista: Sci Rep Año: 2024 Tipo del documento: Article País de afiliación: Australia Pais de publicación: Reino Unido

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