【摘要】 在SARS-CoV的核酸检测方法中,由于普遍缺乏安全、稳定、特异的内对照,而不能对样品处理、反转录、扩增以及定量检测实施全程监控。构建的病毒样核蛋白颗粒内对照,能够对SARS-CoV临床检测实施监控。本文通过克隆1.7Kb大肠杆菌噬菌体MS2的装配蛋白和壳蛋白基因以及SARS冠状病毒经过突变产生的270bp内对照片断,并将这些基因连接到载体pTrc99a上表达,进行纯化、定量分析、RT-PCR检测和稳定性试验。获得SARS内对照病毒样核蛋白颗粒,在人血清和SM缓冲液中37℃稳定性可达到30天,能抵抗核糖核酸酶降解。将内对照颗粒加入临床样本中一同检测,能够对检测的全过程(样品处理、反转录和PCR)有效进行监控,与SARS-CoV没有交叉反应。研究表明制备的该病毒样核蛋白颗粒稳定、安全、可靠,可作为SARS冠状病毒RT-PCR检测、定量分析的有效内对照参考品。更多还原

【Abstract】 For the detection of SARS–CoV using nucleic-acid based methods, the lack of uniform, safe, stable, and specific internal controls for monitoring the entire process from sample preparation, reverse transcription, to PCR amplification has been a problem influencing the specificity and sensitivity of many home-brew and commercially available such systems. The virus-like particles we produced are comparable in quality to those reported for other viruses, and proved a superior internal control for the detection of SARS-CoV using clinical specimens. A 1.7kb DNA fragment coding for the assembly protein and coat protein of MS2 phage, and a 270bp of SARS-CoV fragment with mutated sequences for the internal control probe were cloned and expressed using prokaryotic system. The particles were subject to real time RT-PCR with clinical SARS specimens. Virus-like particles were stable in human plasma and SM buffer for at least 30 days. RNase protection test indicated that various dilutions of virus-like particles could withstand RNase attack. Tests with clinical SARS specimens showed that the produced virus-like particles could be spiked in test samples to serve as the internal control for monitoring the complete detection process from sample preparation, reverse transcription, to PCR amplification. No cross reactions between the virus-like particles and SARS-CoV samples were found when the specific internal control robe was used. The SARS CoV RNA internal control virus-like particle is a useful, durable, ribonuclease-resistant viral RNA internal control for truly clinical assay.更多还原