Detection of viral DNA by isothermal NASBA amplification and chemiluminescence gene probe hybridization assay in a microfluidic cartridge

Background: Detection of viral nucleic acids in biological samples is most often based on molecular amplification followed by sequence-specific detection of amplicons. Isothermal nucleic acid amplification strategies are particularly suited for their implementation in lab-on-chip devices, owing to the simplicity of the protocol and instrumentation required. Among optical detection strategies, chemiluminescence offers the advantages of high detectability in low volumes and no requirements for lamps, filters, or specific optical arrangements. Recently, on-chip integration of photosensors has been proposed to enable highly sensitive detection of chemiluminescence reactions, thus prompting the development of innovative portable analytical devices with enhanced sensitivity and multiplexed capabilities. Methods: A method for amplification of Parvovirus B19 DNA by isothermal nucleic acid sequence-based amplification (NASBA), followed by on-chip hybridisation and chemiluminescence-based detection of amplicons in a portable microfluidic cartridge with integrated photosensors has been developed. Results: Isothermal NASBA for Parvovirus B19 DNA allowed specific amplification of a genotype consensus target, with a limit of detection threshold of 10 genome copies per reaction, equivalent to 100 IU/mL in serum samples. Amplification was followed by on-chip hybridisation and detection of amplicons in a portable microfluidic cartridge, composed of polydimethylsiloxane microfluidic layer coupled with a glass slide bearing an array of thin film a-Si:H photosensors deposited on one side and streptavidin functionalized surface on the opposite side. Biotin-labelled B19 NASBA amplicons were captured and then detected by means of sequence-specific digoxigenin labelled probes and then hybrids were detected by means of an anti-digoxigenin antibody labelled with horseradish peroxidase and chemiluminescence reaction. A linear correlation was obtained between amount of viral target and light emission intensity, with a limit of quantitation of 100 genome copies per reaction. Conclusion: Isothermal amplification of a target viral DNA and its detection by specific hybridization probes and chemiluminescence in a self-contained, portable microfluidic cartridge, attained an analytical performance comparable to standard amplification techniques and detection formats. Integration of a nucleic acid amplification module with the microfluidic cartridge will lead to the design of a portable analytical device, suited for rapid point-of-care molecular diagnosis of viral infections.