A facile one-step method for cell lysis and DNA extraction of waterborne pathogens using a microchip

摘要: Globally, waterborne organisms are the primary causative agents for transmission of various forms diarrheal diseases. For accurate diagnosis, molecular tools have gained considerable attention in recent past. Molecular require DNA as starting material and hence, a prerequisite is quality integrity DNA. To obtain high rapidly, we fabricated microchip poly(dimethyl siloxane) (PDMS) by soft lithography process. The facilitated in-flow coating chitosan on magnetic nanoparticles, which under external mechanical vibration caused cell lysis released supernatant. was captured nanoparticles owing to its positive charge (chitosan coating). nanoparticle-DNA complex then isolated from matrix using permanent magnet, Further, removal debris, proteins, carbohydrates done wash buffer. extracted pure with absorbance (260/280) ratio 1.77±0.04, compared 1.79±0.03 obtained TRIzol method. complete isolation took ~ 15min against>2h Six gram-negative pathogens were used demonstrate efficacy based extraction assessed amplifying 16S rRNA gene Com1 Com2 universal primers. presence band at 407bp gel electrophoresis confirmed amplified product. image quantification product ImageJ software. Higher regression values better opposed conventional lower (<2%) relative standard deviation data suggested that process reproducible. proved simplicity, rapidity, sensitivity microchip-assisted