临床标本中未知病毒基因芯片检测方法探索

目的探索可用于检测临床标本中未知病毒的基因芯片技术。方法设计合成1～4型登革病毒基因芯片探针，制备成登革病毒基因芯片。提取1～4型登革病毒标准毒株的核酸RNA，以phi29DNA聚合酶结合带标签序列的随机引物进行全基因组扩增，再以Cy3荧光染料标记的标签序列引物进行PCR随机扩增标记，标记产物进一步用登革病毒芯片进行杂交检测，随后用广州白云机场出入境检验检疫局国境口岸收集的登革热病人血清标本对新建立的方法进行验证。结果1～4型登革病毒标准毒株培养液提取的RNA，经扩增、标记及芯片杂交检测，相应探针阵列均可检测到明显杂交信号，探针阳性率均达100％；从3份临床登革热病人血清标本中提取的RNA，同样也可得到明显的杂交信号，而背景干扰信号很低，从相应达到100％探针阳性率的探针阵列可判断，3份病人血清标本中分别为含1、2、3型登革病毒。结论该研究新建立的基因芯片方法，可用于检测临床血清标本中的登革病毒，如果设计更多针对不同病原体的特异性芯片探针，该方法还可用于临床标本中一些未知病原体的鉴定。

DNA microarray for detection of unknown virus in clinical specimens

Objective To develop a DNA microarray for detection of unknown virus in clinical specimens. Methods Probes for dengue virus type 1 -4 were designed and synthesized, and DNA microarray of dengue virus was prepared. After viral RNA of dengue virus type 1 - 4 standard strains were extracted, whole genome amplification was performed by using phi29 DNA polymerase and a random primer contain tag sequence, then the genome was amplified and labeled by random PCR with a Cy3 -conjugated tag sequence primer. The fluoreseein labeled amplicon were subjected to hybridization with the DNA microarray of dengue virus. Whereafter the novel method was verified by using serum samples collected from pateints with dengue fever in Guangzhou Baiyun airport entry and exit inspection and quarantine frontier port. Results Hybridization signals of dengue virus type 1 - 4 standard strains were significantly distinguished, and all of the related strains showed a high probe positive rate of 100%. Similarly, 3 serum samples of dengue virus from clinical patients also had significant hybridization signals with very low back-ground noise. Dengue virus types 1,2 and 3 carried in the 3 serum samples can be clearly identified by the high probe positive rate of 100% related to the microarray. Conclusion The innovative DNA microarray assay can be used to detect dengue virus in clinical specimens. If more specific probes for different species of pathogens are designed and appended to this new DNA microarray, it also can be expanded for unknown pathogens identification in clinical specimens.