麻疹病毒属病毒检测基因芯片的建立  

Microarray for species-specific detection of Morbillivirus

作　　者：朱晓光[1] 李斌[1] 刘冰[1] 杨松涛[1] 高玉伟[1] 王铁成[1] 夏咸柱[1] 王承宇[1] 

机构地区：[1]军事医学科学院军事兽医研究所,吉林长春130062

出　　处：《中国病原生物学杂志》2010年第5期329-331,F0003,共4页Journal of Pathogen Biology

基　　金：军队医药卫生科研基金(No.06G138)

摘　　要：目的建立主要麻疹病毒属病毒(麻疹病毒、犬瘟热病毒、小反刍兽疫病毒、牛瘟病毒、海豹瘟热病毒、海豚瘟热病毒等)种特异性检测基因芯片。方法应用生物信息学方法从GenBank获得麻疹病毒属病毒的全部基因组序列,以Primer Premier5.0软件设计针对上述病毒的寡核苷酸探针并制备基因芯片,以上述6种病毒及犬副流感病毒、VERO细胞及健康犬组织等验证寡核苷酸芯片的特异性,以细胞毒和临床病料检测基因芯片的灵敏度、特异性和重复性,芯片制备后低温放置3、6、12个月后检验其检测稳定性。结果基因芯片检测到麻疹病毒属病毒种特异性信号,无杂信号及交叉信号,芯片方法的灵敏度为10-2TCID50,是PCR的100倍,病料检测与PCR相符率为100%。结论初步建立了麻疹病毒属病毒种特异性基因芯片,该方法灵敏度高,特异性强,适用于麻疹病毒属的流行病学调查和种特异性鉴定。Objective A microarray was established to detect Morbillivirus （measles virus,canine distemper virus,Peste des petits ruminants virus,rinderpest virus,porcine distemper virus,dolphin distemper virus,etc.） at the species level. Methods After sequences were downloaded from GenBank,species-specific probes were designed using software Primer Premier5.0. Then,a microarray was prepared with MG2-610. Samples were amplified by HEX-fluoresced PCR primers and hybridized to a microarray,and hybridization results were scanned and studied by scanner. The sensitivity,specificity,and reproducibility of the microarray was determined with cultured viruses and clinical specimens. The stability of microarray was tested after it was stored for 3,6,9,and 12 months at 4 ℃. Results This microarray was specific for Morbillivirus,which did not cross-react with other viruses and cells. The sensitivity of the gene chip was 10^-2TCID50 which is 100 times higher than that of PCR. Detection of specimens with the microarray was consistent with PCR at a rate of 100%.The detection sensitivity and specificity remained unchanged after the kit was assembled for 12 months. Conclusion This study describes a novel assay for detection of Morbillivirus with a high level of sensitivity and specificity,and this assay is suited to use in epidemiological studies and species-specific detection.

关 键 词：基因芯片 麻疹病毒属 种特异性 检测 

分 类 号：R373.11[医药卫生—病原生物学]