西部马脑炎病毒基因组序列的半套式RT-PCR检测

Detection of the genomic sequence of western equine encephalitis virus by seminested RT-PCR

为进一步提高RT PCR检测西部马脑炎病毒 (WEE)病毒基因组方法的敏感性 ,采用半套式PCR扩增病毒基因组特异序列。首先采用逆转录法将病毒基因组RNA逆转录为cDNA ,然后以此cDNA为模板 ,进行扩增。对扩增后电泳检查无可见DNA条带的产物进行半套式PCR ;与此同时对扩增的循环数、Mg++浓度和退火温度等条件进行了优化 ,以进一步提高扩增的特异性。结果第一轮PCR未扩出特异性片段的WEE病毒稀释度 ,其半套式扩增出特定大小的DNA产物 ;同时优化的条件提高了扩增产物的特异性。扩增产物约为 190bp的单一DNA片段 ,其大小与预期的相一致。结果表明采用半套式RT PCR方法检测WEE病毒的基因组序列的敏感性可提高 10

The improvement of the sensitivity of reverse transcription-PCR(RT-PCR) was carried out by seminested-PCR in detection of genome in Westen in Western equine encephalitis (WEE) virus.Firstly, the virus genomic RNA was transcripted to cDNA by reverse transcription, then the DNA fragment was amplified by PCR with the cDNA as a template.The PCR products, which were not observed by agarose electrophoresis,were amplified again by seminested PCR.Meanwhile,some conditions in test procedures including the number of cycle, Mg ++concentration and annealing temperature were optimized, respectivelly. A single and specific DNA fragment was amplified with the optimal conditions. The size of amplified fragment is ablout 190 bp, which is equal to that of the expected product. The sensitivity of detection of WEE virus genome was enhanced more than 100 times by using seminested PCR method.