A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva.
A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva.
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DOI：
10.1039/c3lc40961a
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发表时间：
2013-04-07
期刊：
影响因子：
6.1
通讯作者：
Ramsey JM
中科院分区：
文献类型：
--
作者：
Oblath EA;Henley WH;Alarie JP;Ramsey JM
描述了一种集DNA提取、扩增和检测于一体的用于鉴定唾液中细菌的微流控芯片。该芯片设计集成了用于DNA提取的单片氧化铝膜（AOM）和用于提取的DNA的实时聚合酶链反应（rtPCR）扩增的七个平行反应威尔斯孔。样品首先被加热以裂解靶生物，然后被添加到芯片中并通过纳米多孔AOM过滤以提取DNA。将PCR试剂加入到每个威尔斯孔中，并对芯片进行热循环。唾液样本中变形链球菌的鉴定与300 fg（100-125拷贝）的甲氧西林敏感金黄色葡萄球菌（MSSA）和甲氧西林耐药链球菌的检测沿着得到证实。金黄色葡萄球菌（MRSA）基因组DNA（gDNA）掺入唾液样品中。通过改变7个反应威尔斯孔中使用的引物和探针，可以在同一样品中同时鉴定多个目标菌种和细菌菌株。在最初的测试中，缓冲液中少至30 fg（8-12个拷贝）的MSSA gDNA已成功扩增并用该装置检测。
A microfluidic chip integrating DNA extraction, amplification, and detection for the identification of bacteria in saliva is described. The chip design integrated a monolithic aluminum oxide membrane (AOM) for DNA extraction with seven parallel reaction wells for real-time polymerase chain reaction (rtPCR) amplification of the extracted DNA. Samples were first heated to lyse target organisms and then added to the chip and filtered through the nanoporous AOM to extract the DNA. PCR reagents were added to each of the wells and the chip was thermocycled. Identification of Streptococcus mutans in a saliva sample is demonstrated along with the detection of 300 fg (100–125 copies) of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) genomic DNA (gDNA) spiked into a saliva sample. Multiple target species and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests, as little as 30 fg (8–12 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device.