ABSTRACT
Portable point-of-care devices for pathogen detection require easy, minimal and user-friendly handling steps and need to have the same diagnostic performance compared to centralized laboratories. In this work we present a fully automated sample-to-answer detection of influenza A H3N2 virus in a centrifugal LabDisk with complete prestorage of reagents. Thus, the initial supply of the sample remains the only manual handling step. The self-contained LabDisk automates by centrifugal microfluidics all necessary process chains for PCR-based pathogen detection: pathogen lysis, magnetic bead based nucleic acid extraction, aliquoting of the eluate into 8 reaction cavities, and real-time reverse transcription polymerase chain reaction (RT-PCR). Prestored reagents comprise air dried specific primers and fluorescence probes, lyophilized RT-PCR mastermix and stick-packaged liquid reagents for nucleic acid extraction. Employing two different release frequencies for the stick-packaged liquid reagents enables on-demand release of highly wetting extraction buffers, such as sequential release of lysis and binding buffer. Microfluidic process-flow was successful in 54 out of 55 tested LabDisks. We demonstrate successful detection of the respiratory pathogen influenza A H3N2 virus in a total of 18 LabDisks with sample concentrations down to 2.39 × 10(4) viral RNA copies per ml, which is in the range of clinical relevance. Furthermore, we detected RNA bacteriophage MS2 acting as internal control in 3 LabDisks with a sample concentration down to 75 plaque forming units (pfu) per ml. All experiments were applied in a 2 kg portable, laptop controlled point-of-care device. The turnaround time of the complete analysis from sample-to-answer was less than 3.5 hours.
Subject(s)
Indicators and Reagents/chemistry , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/isolation & purification , Microfluidic Analytical Techniques , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction , Microfluidic Analytical Techniques/instrumentation , Point-of-Care Systems , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/instrumentationABSTRACT
A composite sorbent based on porous glass beads modified with thin polyaniline coating was prepared by precipitating aniline polymerization in the presence of carrier particles. It was shown that the modification ensures the uniform coating of the inner surface of the carrier pores with the polymer layer approximately 70 A thick. It was shown that the resulting material retains the initial porosity of the carrier and is selective in the separation of nucleic acids and proteins. The polyaniline-coated sorbents were shown to be efficient for both the preparative DNA isolation from bacterial lysates and for analytical purposes, in particular, for studying DNA fragmentation during apoptosis proceeding under UV irradiation of cell lysates of colon carcinoma. The morphological and chromatographic characteristics of the new sorbent were demonstrated to be similar to those of the polyfluorobutadiene sorbent.
Subject(s)
Aniline Compounds/chemistry , Biochemistry/methods , DNA/isolation & purification , Silicon Dioxide/chemistry , Apoptosis/genetics , Apoptosis/radiation effects , Carcinoma/genetics , Carcinoma/pathology , Chromatography/methods , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , DNA/chemistry , DNA, Bacterial/isolation & purification , DNA, Neoplasm/isolation & purification , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Humans , Ultraviolet RaysABSTRACT
A new method for the identification of point mutations is proposed. The method is based on ligase chain reaction (LCR) and it includes a procedure for correction of ligation by Cleavase. Reaction products are detected by a colorimetric method after adsorption of the resulting DNA duplexes to the solid phase. One strand of LCR products carries biotin to be bound on a streptavidin-coated microwell. Another strand contains a single-stranded region that is to be coupled with an oligonucleotide carrying a substrate for colorimetric detection. The suggested method has two advantages: (i) use of Cleavase increases the accuracy of ligation and (ii) a template independent ligation does not occur in LCR due to a special design of primers.
Subject(s)
DNA Ligases/metabolism , DNA Mutational Analysis , Endodeoxyribonucleases/metabolism , Genetic Techniques , Point Mutation , Base Sequence , Biotin/metabolism , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Mutation , Oligonucleotides/chemistry , Sequence Homology, Nucleic AcidABSTRACT
RNA isolation from purified cucumber mosaic virus, CMV-U strain and cDNA synthesis was carried out. The coat protein gene (RNA4) region was amplified selectively by polymerase chain reaction (PCR) with CMV RNA4-specific primers. Double-stranded cDNA was cloned in PRT103 vector at Xho1/Kpn1 site and about 1kb insert obtained. The insert was partly sequenced which showed 50% sequence homology with CMV-Q, C and WL strains.
