全基因组扩增策略在基因芯片分析SARS冠状病毒实验中的应用

Strategy of Whole Genome Amplification in Analysis of SARS Coronavirus with Microarray

针对SARS冠状病毒的分子生物学检测是控制SARS流行的关键环节。为评价全基因组扩增对SARS微量样本检测的影响 ,采用 6 mer随机引物反转录 ,用加接头的随机引物合成第二链 ,再以接头序列为引物扩增并掺入荧光标记 ,最后与带有 70 mer探针的基因芯片杂交。此非特异方法基本覆盖了样本中的全部DNA ,结果发现SARS冠状病毒全基因组的扩增效果对基因芯片杂交结果的均匀性有较大影响 ,PCR循环次数增多会导致扩增均匀性的降低。分析了不同的引物对全基因组扩增均匀性的影响 ,探讨了全基因组扩增策略的缺陷。

SARS Coronavirus detection by molecular biological method is the key step to control the prevalence of SARS.In order to evaluate the influence of whole genome amplification on SARS CoV detection in minimum amount of specimen,the virus whole genome was amplified by a random primer method and combined it with microarray hybridization.The efficiency of the SARS whole genome amplification had prominent influence on the detection result.The fidelity in SARS whole genome amplification and the amplification effect by different primers were analyzed.The disadvantages of the strategy of whole genome amplification were also discussed.