腹泻病毒化学发光基因芯片检测方法的建立和验证

Development and validation of diarrhea virus by chemiluminescence DNA microarray

目的建立一种化学发光基因芯片检测方法,实现7种腹泻病毒,A组轮状病毒、B组轮状病毒、Ⅰ型诺如病毒、Ⅱ型诺如病毒、札如病毒、星状病毒和肠道腺病毒的快速、准确检测。方法选择7种病毒特异性基因的保守区,设计引物与探针,制备寡核苷酸基因芯片。将多重实时荧光PCR(RT-PCR)扩增产物与带有特异性探针的芯片杂交,经洗涤、化学发光检测后进行结果分析。在优化的RT-PCR体系、杂交条件和化学发光检测条件下,评价芯片的灵敏度、重复性和特异性。结果研制的基因芯片具有良好的特异性和灵敏度,检测体外转录RNA参考品的最低检测限为3×103拷贝/反应,检测临床样本的灵敏度为95.2%、特异性为92.1%、符合率为95.1%。结论建立了一种基于化学发光基因芯片的腹泻病毒检测方法,此法能快速、灵敏、特异地检测和鉴别7种腹泻病毒,具有较好的应用前景。

Objective To develop a chemiluminescence（ CL） imaging DNA microarray method for quick and accurate detection of seven viruses associated with acute gastroenteritis,namely,Rotavirus A,Rotavirus B,Norovirus G Ⅰ,Norovirus GⅡ,Sapovirus,Astrovirus and enteric adenovirus. Methods Primers and probes were designed based on the specific sequence in conserved regions of seven diarrhea virus genomes to prepare the oligonucleotide microarray. The multiplex RT-PCR amplification products were hybridized with the microarray of specific probes and with the microarray that was scanned after washing and chemiluminescence coloration before the microarray was scanned to identify the type of virus.After the optimization of the RT-PCR system,hybridization,and visualization,the specificity,sensitivity and reproducibility of the chip were evaluated. Results The microarray demonstrated an outstanding sensitivity and specificity. The sensitivity rate and specificity rate of clinical samples detection were 95. 2% and 92. 1% respectively,while the accuracy rate was95. 1%. The minimum detection limit of in vitro transcribed RNAs was 3 × 10^3 copies / reaction. Conclusion A detection method for diarrhea viruses is established based on chemiluminescence DNA microarray,which can detect and identify seven diarrhea viruses quickly,sensitively and specifically,with good prospects of application.