A facile one-step method for cell lysis and DNA extraction of waterborne pathogens using a microchip
A facile one-step method for cell lysis and DNA extraction of waterborne pathogens using a microchip
复制标题
DOI：
10.1016/j.bios.2017.07.040
复制
发表时间：
2018-01-15
影响因子：
12.6
通讯作者：
Bodas, Dhananjay
中科院分区：
文献类型：
--
作者：
Kamat, Vivek;Pandey, Sulaxna;Bodas, Dhananjay
在全球范围内，水媒生物是传播各种形式疟疾的主要病原体。为了准确诊断，分子工具在最近的过去得到了相当大的关注。分子工具需要DNA作为诊断的起始材料，因此，先决条件是DNA的质量和完整性。为了快速获得高质量的DNA，我们采用软光刻技术在聚二甲基硅氧烷（PDMS）中制作了微芯片。该微芯片促进壳聚糖在磁性纳米颗粒上的流动涂层，其在外部机械振动下引起细胞裂解并在上清液中释放DNA。释放的DNA因其带正电荷（壳聚糖涂层）而被纳米颗粒捕获。然后使用永磁体从流入基质中分离磁性纳米颗粒-DNA复合物。此外，使用洗涤缓冲液去除细胞碎片、蛋白质和碳水化合物。使用微芯片提取的DNA是纯的，吸光度（260/280）比为1.77 +/- 0.04，而TRIzol方法获得的吸光度（260/280）比为1.79 +/- 0.03。使用微芯片完全分离DNA需要15分钟，而使用TRIzol方法需要> 2小时。使用六种革兰氏阴性水性病原体来证明基于微芯片的DNA提取过程的功效。通过使用Coml和Com 2通用引物扩增16 S rRNA基因来评估分离的DNA的完整性。凝胶电泳在407 bp处出现一条带，证实了扩增产物。此外，使用ImageJ软件将凝胶图像用于扩增产物的定量。与传统方法相比，使用微芯片获得的更高回归值证实了提取的DNA的质量和完整性更好。从数据中获得的较低（< 2%）的相对标准偏差值表明，微芯片工艺是可重现的。所获得的DNA的质量和完整性证明了微芯片辅助DNA提取过程的简单、快速和灵敏度。
Globally, waterborne organisms are the primary causative agents for the transmission of various forms of diarrheal diseases. For accurate diagnosis, molecular tools have gained considerable attention in the recent past. Molecular tools require DNA as the starting material for diagnosis, and hence, a prerequisite is the quality and integrity of DNA. To obtain high quality DNA rapidly, we have fabricated a microchip in poly(dimethyl siloxane) (PDMS) by soft lithography process. The microchip facilitated in-flow coating of chitosan on the magnetic nanoparticles, which under external mechanical vibration caused cell lysis and released DNA in the supernatant. The released DNA was captured by the nanoparticles owing to its positive charge (chitosan coating). The magnetic nanoparticle-DNA complex was then isolated from the in-flow matrix using permanent magnet, Further, removal of the cell debris, proteins, and carbohydrates was done using wash buffer. DNA extracted using the microchip was pure with absorbance (260/280) ratio of 1.77 +/- 0.04, as compared to 1.79 +/- 0.03 obtained by TRIzol method. The complete isolation of the DNA using the microchip took 15 min as against > 2 h with a TRIzol method. Six gram-negative waterborne pathogens were used to demonstrate the efficacy of the microchip based DNA extraction process. The integrity of the isolated DNA was assessed by amplifying the 16S rRNA gene using Coml and Com2 universal primers. The presence of a band at 407 bp on gel electrophoresis confirmed the amplified product. Further, the gel image was used for quantification of the amplified product using ImageJ software. Higher regression values obtained using microchip confirmed better quality and integrity of the extracted DNA as opposed to the conventional method. The lower (< 2%) relative standard deviation values obtained from the data suggested that the microchip process was reproducible. The quality and integrity of the obtained DNA proved the simplicity, rapidity, and sensitivity of the microchip-assisted DNA extraction process.