含内质控多重荧光RT-PCR同时检测麻疹病毒和风疹病毒的研究

Simultaneous Detection of Measles and Rubella Virus by Multiplex Real-time RT-PCR with an Internal Control

由于从临床症状上难以区分麻疹病毒或风疹病毒引起的人群急性发热出疹性传染病,故而分别设计针对麻疹病毒和风疹病毒的特异性引物和荧光标记探针,建立含内质控的多重荧光RT-PCR以同时检测麻疹病毒和风疹病毒的核酸。结果表明:建立的多重荧光RT-PCR方法特异性好,试验验证没有出现假阳性和假阴性现象;该方法的灵敏度高,对麻疹病毒和风疹病毒的最低检出限分别达到0.1TCID50/mL和1TCID50/mL,可从疑似患者咽拭子等标本中直接检测到病毒核酸;同时该方法具有良好的稳定性,当分别用0.1～103TCID50/mL麻疹和风疹病毒的样本进行重复试验时,其CT值变异系数均在0.9%以下。此外,以人核糖核酸酶P(RNaseP)作为内质控,可以有效监控临床标本的采样质量及因操作失误等所致的假阴性结果出现。本方法尤其适用于疑似麻疹或风疹暴发疫情的临床快速诊断。

Measles and rubella virus cause fever rash diseases that are uneasy to differentiate clinically from each other. Specific primers and fluorescence-labeled probes were designed,and a multiplex Real-time RT-PCR with an internal control was developed to simultaneously identify the measles and rubella virus. The multiplex Real-time RT-PCR assay was specific and no false positive or false negative results were found. The sensitivity of the assay was 0.1TCID50/mL and 1TCID50/mL for measles and rubella virus respectively. Analysis with 0.1-10^3TCID50/mL measles or rubella virus samples demonstrated high validity and reproducibility with the coefficient of variability（CV） of below 0.9% for both measles and rubella virus. Using ribonuclease P （RNase P） as internal false negative control,the developed multiplex Real-time RT-PCR assay is suitable for rapid clinical diagnosis of measles and rubella virus.