A Syringe-Based and Centrifugation-Free DNA Extraction Procedure for the Rapid Detection of Bacteria

Abstract

Several bacteria are known to cause food poisoning; therefore, diagnostic systems that detect bacteria are essential. Nucleic acid-based testing methods that involve polymerase chain reaction (PCR) amplification are of great interest due to their high sensitivity and specificity. Herein, we developed a syringe-based one-step DNA extraction device that streamlines the extraction of genomic DNA (gDNA) from bacteria within 2 min, enabling versatile application of nucleic acid-based testing in the field. Notably, the bolt-nut structured case coupled with the syringe enables control of the volume of solution dispensed for enabling DNA extraction without the need for bulky centrifuge equipment. Using the proposed system, the gDNA of a model bacterium, Escherichia coli, was extracted at a good quantity and quality and amplified via PCR. The DNA extracted was comparable to that extracted via a centrifugation-based procedure. In addition, bacteria that were artificially spiked in common samples, including a work cloth, a work bench, and meat, were successfully detected with high accuracy.

Amplificación directa de ADN de Bordetella pertussis purificado de hisopados nasofaríngeos por un método de bajo costo, rápido (60-segundos) y libre de equipos

“…Aunque la metodología NPM ha mostrado un buen rendimiento en la obtención de ADN en un tiempo promedio de 10 min, requiere del uso de equipos y la preparación relativamente compleja de nanopartículas. Dispositivos libres de centrifugación han sido desarrollados para la extracción rápida de ADN en 2 min con rendimiento comparable al purificado por un kit comercial (29) ; sin embargo, no están acoplados a una amplificación directa por PCR o LAMP y se limitan al análisis de una sola muestra. Hasta la fecha, no existen reportes de métodos rápidos de purificación y amplificación directa de ADN de B. pertussis de hisopados nasofaríngeos; por lo tanto, se propone en el presente estudio una nueva alternativa simple y rápida de purificación de ácidos nucleicos en este tipo de muestras clínicas.…”

Rapid on-site nucleic acid testing: On-chip sample preparation, amplification, and detection, and their integration into all-in-one systems

“…A silica membrane can bind with DNA at a high salt concentration. Yoon et al (2021) designed a syringe-based DNA extraction device that uses a silica membrane to separate out target DNA. In some cases, a commercially available filter is externally connected to the chip; this approach makes for an easy design process, but potentially presents problems with leaking ( Ryzhkov et al, 2020 ).…”

Advances in Pretreatment Methods for Free Nucleic Acid Removal in Wastewater Samples: Enhancing Accuracy in Pathogenic Detection and Future Directions

“…Following cell lysis, the sample solution contains a mixture of nucleic acids, including both the genetic material of the target pathogen and interfering free nucleic acids. To selectively isolate the nucleic acids from other components, various extraction methods, such as spin column-based extraction, magnetic bead-based extraction, or organic extraction, have been applied [26][27][28]. After pretreatment and extraction, the concentration of target pathogen's genetic material can be detected using diverse methods, such as sequencing and real-time PCR.…”