RESUMEN
BACKGROUND: At present, the process of inspection and quarantine starts with sampling at the customs port, continues with transporting the samples to the central laboratory for inspection experiments, and ends with the inspected results being fed back to the port. This process had the risks of degradation of biological samples and generation of pathogenic microorganisms and did not meet the rapid on-site detection demand because it took a rather long time. Therefore, it is urgently needed to develop a rapid and high-throughput detection assay of pathogenic microorganisms at the customs port. The aim of this study was to develop a microfluidic chip to rapidly detect swine pathogenic microorganisms with high-throughput and higher accuracy. Moreover, this chip will decrease the risk of spreading infection during transportation. RESULTS: A series of experiments were performed to establish a microfluidic chip. The resulting data showed that the positive nucleic acid of four swine viruses were detected by using a portable and rapid microfluidic PCR system, which could achieve a on-site real-time quantitative PCR detection. Furthermore, the detection results of eight clinical samples were obtained within an hour. The lowest concentration that amplified of this microfluidic PCR detection system was as low as 1 copies/µL. The results showed that the high specificity of this chip system in disease detection played an important role in customs inspection and quarantine during customs clearance. CONCLUSION: The microfluidic PCR detection system established in this study could meet the requirement for rapid detection of samples at the customs port. This chip could avoid the risky process of transporting the samples from the sampling site to the testing lab, and drastically reduce the inspection cycle. Moreover, it would enable parallel inspections on one chip, which greatly raised the efficiency of inspection.
Asunto(s)
Dispositivos Laboratorio en un Chip/veterinaria , Reacción en Cadena de la Polimerasa Multiplex/veterinaria , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Enfermedades de los Porcinos/virología , Animales , Circovirus/genética , Circovirus/aislamiento & purificación , Herpesvirus Suido 1/genética , Herpesvirus Suido 1/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento , Dispositivos Laboratorio en un Chip/virología , Microfluídica/instrumentación , Reacción en Cadena de la Polimerasa Multiplex/métodos , Virus de la Diarrea Epidémica Porcina/genética , Virus de la Diarrea Epidémica Porcina/aislamiento & purificación , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Porcinos , Enfermedades de los Porcinos/diagnósticoRESUMEN
CONTEXT.: Zika virus (ZIKV) infection, primarily transmitted by mosquitoes, causes various neurologic disorders. To differentiate ZIKV from other arboviruses, such as dengue, chikungunya, and yellow fever viruses, a highly specific, sensitive, and automated detection system is needed for point-of-care (POC) settings. OBJECTIVE.: To detect ZIKV at POC settings, we have developed a fully automated lab-on-a-chip microfluidic platform for rapid disease detection by using reverse transcription loop-mediated isothermal amplification. DESIGN.: The developed setup consists of a microfluidic chip, a platform for magnetic actuation, and a heater along with the sensor to precisely control the temperature for the target amplification. The platform accurately controls the movement of the magnetic beads that enable the isolation and purification of the target nucleotides adhered to their surface for the amplification and disease detection on the microfluidic chip. RESULTS.: Within 40 minutes, change in color due to the presence of ZIKV amplicons was visually observed with the spiked plasma samples in the end point analysis. Also, we have accurately and specifically identified ZIKV in a small number of de-identified clinical samples. CONCLUSIONS.: All-inclusive, the developed fully automated POC ZIKV diagnostic chip is rapid, simple, easy to use, inexpensive, and suitable for the areas where facilities are limited.
Asunto(s)
Dispositivos Laboratorio en un Chip/normas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Pruebas en el Punto de Atención/normas , Infección por el Virus Zika/diagnóstico , Virus Zika/genética , Animales , Humanos , Dispositivos Laboratorio en un Chip/estadística & datos numéricos , Dispositivos Laboratorio en un Chip/virología , Sistemas de Atención de Punto , Pruebas en el Punto de Atención/estadística & datos numéricos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Virus Zika/fisiología , Infección por el Virus Zika/virologíaRESUMEN
BACKGROUND: PPV is one of the most important pathogens causing porcine reproductive disorder. It has been shown in clinical cases to be a commonly mixed infection with other important swine diseases which can aggravate the severity of the disease and bring serious economic losses to the pig industry. Serological methods, such as hemagglutination inhibition assays (HAI), serum neutralization (SN), and the modified direct complement-fixation (MDCF) test were utilized earlier, whereas the enzyme-linked immunosorbent assay (ELISA) is the most frequently applied assay to detect PPV-specific antibodies. RESULTS: We establish the visible protein chip and the cyanine dye 3 (Cy3)-labeled protein chip to detect the clinical serum from pigs. In this study, the recombinant protein VP2 of PPV was expressed in E.coli, purified with nickel magnetic beads, and then printed onto epoxy-coated glass slides for preparation of the protein chip. After a series of experiments, the conditions of antigen protein concentration, incubation time of primary antibody or secondary antibody, and optimal serum dilution fold were optimized, resulting in a successful visible protein chip and Cy3-labeled protein chip. The results showed that the positive serum, diluted up to 6000-fold, can be detected by the visible protein chip, and the positive serum, diluted up to 12,800-fold, can be detected by the Cy3-labeled protein chip, suggesting the high sensitivity of these protein chips. Moreover, the positive detection ratio, sensitivity, and specificity of these two kinds of protein chips were higher than those of commercial ELISA antibody detection kits. CONCLUSION: Overall, these two protein chips can be used to rapidly diagnose clinical samples with high throughput.
Asunto(s)
Anticuerpos Antivirales/sangre , Dispositivos Laboratorio en un Chip/veterinaria , Infecciones por Parvoviridae/veterinaria , Parvovirus Porcino/aislamiento & purificación , Enfermedades de los Porcinos/virología , Animales , Dispositivos Laboratorio en un Chip/virología , Infecciones por Parvoviridae/diagnóstico , Infecciones por Parvoviridae/virología , Sensibilidad y Especificidad , Porcinos , Enfermedades de los Porcinos/sangre , Enfermedades de los Porcinos/diagnósticoRESUMEN
We have developed a high-throughput, microfluidics-based platform to perform kinetic analysis of viral infections in individual cells. We have analyzed thousands of individual poliovirus infections while varying experimental parameters, including multiplicity of infection, cell cycle, viral genotype, and presence of a drug. We make several unexpected observations masked by population-based experiments: (1) viral and cellular factors contribute uniquely and independently to viral infection kinetics; (2) cellular factors cause wide variation in replication start times; and (3) infections frequently begin later and replication occurs faster than predicted by population measurements. We show that mutational load impairs interaction of the viral population with the host, delaying replication start times and explaining the attenuated phenotype of a mutator virus. We show that an antiviral drug can selectively extinguish the most-fit members of the viral population. Single-cell virology facilitates discovery and characterization of virulence determinants and elucidation of mechanisms of drug action eluded by population methods.