含非竞争性内标四重荧光RT-PCR方法检测手足口肠道病毒方法的研究

旨在了解手足口病的流行和感染情况,并进行快速准确的检测。建立了含非竞争性内标的同时检测肠道病毒通用型、肠道病毒EV71型及柯萨奇病毒CA16型的四重荧光RT-PCR方法,对该方法的特异性、灵敏度等进行评价,并对多份临床样本进行应用检测。结果表明,该检测方法特异性强,对肠道病毒及其他人类非肠道病毒进行检测,显示了良好的特异性;该检测方法对EV71型和CA16型的检测灵敏度分别达到31.25 TCID50和1.25×102TCID50;将浓度为1×104TCID50及5×102TCID50的EV71样本进行重复性试验,其变异系数均小于1.5%;将浓度为5×102-5×105TCID50的EV71和CA16样本进行线性试验,其相关系数R2值在0.982-0.998之间。采用本研究建立的方法检测40份疑似临床样本,最后检出31份肠道病毒阳性样本,其中8例EV71型阳性,13例CA16阳性。另外,试验数据表明,内标对监控PCR抑制物的存在具有重要作用。本方法能同时快速检测所有肠道病毒并进行EV71型及CA16型的分型,并且灵敏度高、特异性好、扩增效率高,由于加入了内标,能有效地监控假阴性的出现,适合于手足口病的临床检测。

Multiplex Real-time PCR Detection of Enteroviruses of HFMD with a Non-competent Internal Control

It was to rapidly and effectively identify and genotype the HFMD enterovirus simultaneously.An up to 4-plex real-time PCR with a non-competent internal control(IC)was developed.The specificity,sensitivity,reproducibility and linearity of the real time RT-PCR assay were estimated and about 40 clinical samples were tested.The results showed good specificity for the selected virus.The assay met the sensitivity of 31.25 TCID50 sample for EV71 and 1.25×102 TCID50 sample for CA16.Analysis with 1×104 TCID50 and 5×102 TCID50 EV71 samples demonstrated high reproducibility with a coefficient of variation(CV)below 1.5%.The linearity analysis with 5×102-5×105 TCID50 for EV71 and CA16 show a good correlation and amplification efficiency.About 40 clinical samples were detected by Real-time PCR,The results showed that the positive ratio was 77.5%,higher than common assay.Moreover,the result indicated that there was PCR inhibition in some samples.The inhibition in these samples showed the importance of IC when PCR was used to detect the RNA of EV71,CA16 or other enterovirus.As a result of its high specificity,sensitivity,linearity and avoiding false negative results by using an internal control,the assay is suitable for rapid clinical diagnosis of human enterovirus and genotyping of EV71 and CA16.