Isothermal solid-phase recombinase polymerase amplification on microfluidic digital versatile discs (DVDs)

Abstract

A new advancement in massive DNA-based screening in limited-resource settings is demonstrated through the incorporation of easy-to-fabricate microfluidic chambers on digital versatile discs (DVDs) to perform isothermal recombinase polymerase amplification (RPA) in a microarray format. Standard un-modified DVD discs and commercial drives are used for the low-cost detection method. DNA primers were printed in a microarray format on the polycarbonate surfaces of DVDs with integrated control spots to guarantee the absence of false-negatives and false-positives. The solid-phase amplification assay, including the washing protocols and development reaction, was performed by the dispensation of solutions through the inlet and by controlling the flow-movement by DVD drive centrifugation. The final disc with reaction products was inserted into a DVD player and microarray images were captured and automatically processed. This simple approach was applied for the screening of genetically modified organisms (GMOs) in food samples. The limit of detection was 7 mg g À1 , which is well below the EU regulation limit for GMOs in food products. Therefore, the only required materials for food safety monitoring were standard store-bought DVDs, plastic chambers, tips, pipettes, an oven, and a standard DVD drive. The proposed strategy allows an integrated microarray system with low manipulation, reduced sample volume, and portability, which are beneficial for low-resource settings.

Recombinase polymerase amplification: Basics, applications and recent advances

“…Other reports detail pseudomultiplexing platforms through parallelised single reactions, using foil based centrifugal microfluidic cartridges with stored reagents [49,58], digital versatile discs (DVD) [41,42,44], vacuum degassed microfluidic cartridges [59] or polylactic acid/polycarbonate chips [6].…”

“…DVDs and low reflectivity DVDs [41] are suitable substrates for the immobilisation of primers for solid phase or bridge amplification, facilitating multiplexing through parallelisation in individual reactors of the DVD. Once amplification is achieved a DVD reader can be used to read out the results in reflection [42] or transmission mode [44].…”

“…Once amplification is achieved a DVD reader can be used to read out the results in reflection [42] or transmission mode [44]. Additionally, the DVD drives provide centrifugal force to actuate microfluidics for aliquoting and mixing [41].…”

“…Other approaches such as lab in a suitcase [62], combine all the components needed to perform RPA in situ, using a portable fluorometer for the amplification read-out and portable solar panels and batteries as power sources. Other approaches such as electrochemical solid phase amplification [30] or solid phase amplification on DVDs [41] have potential for multiplexed detection of target at the point of need, but further research is required to reduce the number of steps or to automate the whole process.…”

Detection methods for centrifugal microfluidic platforms

“…Scattering of the laser due to the presence of particles/cells results in readout errors, which are counted and correlated with the concentration of particles/cells. An integrated system for isothermal DNA amplification to detect genetically modified organisms (GMOs) in food samples has been developed by Tortajada-Genaro et al (Tortajada-Genaro et al, 2015). Detection arrays are printed on standard DVDs and subsequently a layer containing the microfluidic network for sample processing is reversibly bonded to the disc.…”