RESUMEN
The present report introduces the smartphone as a simple, low-cost detector/imager for chemiluminometric hybridization assays and quantitative competitive polymerase chain reaction (QCPCR). In QCPCR the amplification products from the target and the competitor DNA have identical sizes but differ in a short sequence flanked by the primers. The products are hybridized with their cognate oligonucleotide probes, captured on microtiter wells and detected via an enzyme-catalyzed chemiluminogenic reaction using the smartphone as a detector/imager. We provide, for the first time, data on: (a) the detectability, analytical range and reproducibility of smartphone-based chemiluminometric hybridization assays of double stranded amplification products, (b) the comparison of smartphone-based detection with a conventional digital camera and a luminometer, and (c) the detectability, analytical range and reproducibility of smartphone-based QCPCR in terms of the number of copies of input target sequences in the sample prior to amplification. The limits of detection of the DNA hybridization assay based on the smartphone, digital camera and luminometer were 1.6, 2.4 and 1â¯pmolâ¯L-1. Smartphone-based QCPCR showed an analytical range from 137 to 9â¯×â¯105 copies of target DNA.
Asunto(s)
Mediciones Luminiscentes/instrumentación , Hibridación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/instrumentación , Teléfono Inteligente , Sondas de Oligonucleótidos/química , Sondas de Oligonucleótidos/genética , Reproducibilidad de los ResultadosRESUMEN
Nucleic acid-based tests have a profound impact in every medical discipline. Because multigene tests offer higher diagnostic accuracy and lower overall cost than single assays, they are especially useful for diseases, like prostate cancer, that present variability at the molecular level and diversity of available therapeutic interventions. We have developed a quantitative competitive PCR for an eight-gene panel, related to prostate cancer, that includes five genes of the human tissue kallikrein family (KLKs), prostate-specific membrane antigen (PSMA), prostate cancer antigen 3 (PCA3), and HPRT1 as a reference gene. Using PCR as a synthetic tool, a competitor was prepared for each target sequence containing the same primer binding sites as the target but differing in a short segment to enable discrimination by hybridization. The assay involves multiplex amplification of targets and competitors followed by a multiplex hybridization assay for the 16 amplification products. The assay was performed on optically encoded microspheres with oligonucleotide probes attached to their surface. The microspheres were analyzed rapidly (1 min) by flow cytometry. The signal ratio of the target and cognate competitor is a function of the target copy number in the sample prior to amplification. The multiplexing potential of the proposed method is much higher than real-time PCR and other end-point methods since there are 100 sets of commercially available microspheres.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Reacción en Cadena de la Polimerasa/métodos , Neoplasias de la Próstata/genética , Antígenos de Neoplasias/genética , Antígenos de Superficie/genética , Línea Celular Tumoral , Citometría de Flujo/métodos , Colorantes Fluorescentes/análisis , Glutamato Carboxipeptidasa II/genética , Humanos , Calicreínas/genética , MasculinoRESUMEN
MicroRNAs (miRNAs) are single-stranded noncoding RNA molecules that act as key regulators of mRNA expression and are emerging biomarkers for disease. Their small size (18-25 nt) presents challenges to molecular recognition, labeling, and signal generation. Recent research activity in this field has aimed at the development of methods for miRNA quantification that combine high detectability, broad dynamic range, practicality, multiplexity, and low cost for prospective applications in diagnostic laboratories. This review article covers the most recent advances in microRNA analysis.
