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为了探索猪繁殖与呼吸综合征病毒(PRRSV)细胞受体CD163在宿主感染PRRSV过程中的表达情况,根据CD163的序列在其保守区设计了1对特异性引物,基于SYBR GreenⅠ荧光染料建立了一步法检测CD163的实时荧光定量RT-PCR(real time RT-PCR)法,并对该方法进行了特异性、敏感性、重复性试验及样品检测试验。结果表明:建立的方法具有较高的特异性,PRRSV的其他几个受体(CD151、HS)均为阴性反应;对CD163的检测下线为10个拷贝数的模板RNA,比常规RT-PCR方法的灵敏度高10倍;对样品进行3次组内和组间重复试验,变异系数(CV)<2%,具有较好的重复性和稳定性;利用该方法对PRRSV感染Marc-145细胞后不同时间段CD163的转录水平进行检测,结果显示CD163在48小时时的mRNA含量明显高于其他各时间段。说明所建立的针对CD163的检测方法完全可以应用于临床样品的检测。
In order to explore the expression of porcine reproductive and respiratory syndrome virus (PRRSV) cell receptor CD163 in host-infected PRRSV, a pair of specific primers was designed according to the sequence of CD163 in its conserved region. Based on the SYBR GreenⅠ fluorescent dye, Method was used to detect the CD163 real-time RT-PCR method. The specificity, sensitivity, repeatability and sample test of this method were also studied. The results showed that the established method was highly specific, and several other PRRSV receptor (CD151, HS) were negative reaction; the detection of CD163 off-line for the 10 copy number of template RNA, compared with conventional RT-PCR The sensitivity of the method was 10-fold higher. The sample was repeated 3 times within and between groups and the coefficient of variation (CV) was less than 2%. The method had good repeatability and stability. After the infection of Marc-145 cells by PRRSV The results showed that the mRNA level of CD163 at 48 hours was significantly higher than that of other time periods. Indicating that the established detection method for CD163 can be completely applied to the detection of clinical samples.