基于DNA杂交CD式微流控芯片筛查苯丙酮尿症方法的建立

Development of a compact disk type microfluidic chip based on DNA hybridization for phenylketonuria screening

目的 建立一种基于往复流的DNA杂交CD式微流控芯片技术,用于PKU基因突变快速筛查.方法 设计并加工一种具有12个微通道的CD式微流控芯片,芯片采用PDMS-玻璃双层结构,分别含有微通道和水凝胶结合探针区.探针区包括R243Q、V245V和空白对照.将PAH外显子7扩增产物加入芯片的进样孔,在离心力的作用下进入杂交微通道区.芯片在离心机中交替旋转或暂停从而进行杂交反应.杂交后将芯片置于荧光显微镜下观察结果并分析荧光信号.评价该方法检测特异性、检测限及重复性.对30例疑似PKU孕妇DNA标本进行检测,并将检测结果与测序比较.结果 利用基于往复流的DNA杂交CD式微流控芯片只需1.5μl样品,杂交时间为15 min,杂交检测限为0.7 ng/μl.对于PKU患者组与健康对照组,芯片检测结果与测序结果一致.挑选不同批次的5张芯片及每张芯片内5个微通道重复检测同一V245V突变PKU患者DNA标本,结果均为阳性,说明重复性较好.对30例疑似PKU孕妇标本进行检测,筛查出4例携带V245V突变,1例携带R243Q突变.结论 建立了一种基于往复流的DNA杂交CD式微流控芯片检测PKU基因突变的方法,该方法简便、快速、高灵敏,可以用于PKU产前筛查.

Objective To develop a phenylketonuria （PKU） screening method based on a compact disk （CD） type microfluidic chip capable of generating reciprocating flow within the microchannels that facilitate rapid DNA hybridization. Methods This microfluidic device consists of a two-layer structure： a polydimethylsiloxane （PDMS） top layer containing 12 DNA hybridization microchannels, and a bottom glass layer with immobilized hydrogel conjugated DNA arrays. The DNA arrays included R243Q, V245V and the blank control probes. When the CD device was spun, the PCR products were driven into the hybridization channel by centrifugal force. When the rotation of the CD device was stopped, capillary force pulled the PCR products solution to flow back to the channel. After the on-chip hybridization, the hybridization signals were captured on a fluorescence microscope. The specificity, detection limitation and reproducibility of this device were evaluated. Thirty DNA samples from pregnant women with suspected PKU were detected by this device.Then the results were compared with DNA sequencing results. Results With the compact disk type microfluidic chip, the hybridization time could be reduced to 15 min, sample consume could be as low as 1. 5 μl and the detection limitation was 0. 7 ng/μl. With the chip based method, samples of PKU patients and healthy controls were detected and the results were consistent with DNA sequencing results. Five different batches of chips and five micro-channels of each chip were selected to test one PKU patients with V245V mutation. All the results were positive, indicating good reproducibility. Four cases of V245V mutation and 1 case of R243Q mutation were found in 30 suspected PKU carried pregnant women. Conclusion The compact disk microfluidic device has advantages of simple, rapid and highly sensitive, thus is well suited to PKU screening.