摘要
目的建立以特异性荧光探针为特点的TaqMan荧光定量RT-PCR方法,对甲肝病毒核酸进行检测。方法根据GenBank发表的HAV全基因组序列资料分析结果,在其保守区段设计HAV特异性引物和探针,优化引物、探针浓度,与最佳退火温度,并考察检测体系的特异性、敏感性与重复性。结果本研究体系的引物和探针浓度为0.8μmol/L和0.6μmol/L,退火温度为52℃时,具有良好的特异性与敏感性,检测灵敏度可达0.1TCID50。同一样品重复检测3次,Ct值的变异系数均小于5%。结论本研究建立的HAV TaqMan RT-PCR方法特异、敏感、快速且有效减少交叉污染的概率,为临床及水产品中的甲肝病毒的早期快速检测提供了一种新方法。
Objective To establish a real- time PCR assay based on TaqMan chemistry for detection of Hepatitis A Virus RNA. Methods Sixteen HAV genomie sequences were analyzed and HAV specific primers and probe in 460 - 533 bp by primer Express 2.0 Primer and probe ratio were optimized.The best annealing temperature, specificity and maintainability analysis tests were performed by other flavivirus of encephalitis associated virus.Sensitive and stability analysis was tested by tittered virus stock. Results Ratio of primer and probe is 0.8 μmol/L and 0.6 μmol/L.The best annealing temperature is 52 ℃. Test showed that the primers and probe were highly conservative and specific.The low detecting limit was 0.1 TCID50. Stability test showed the co-efficient variables was all less than 5% in 3 different samples.Results showed that TaqMan PCR for HAV is more sensitive, easier and faster to perform the traditional virus isolation and identification. Conclusion This real - time RT - PCR TaqMan - based assay was a specific, sensitive and quick tool suitable for early and quick detection of HAV in clinical and aquatic products.
出处
《中国预防医学杂志》
CAS
2007年第3期229-232,共4页
Chinese Preventive Medicine
