SARS病毒核酸检测中不同引物对灵敏度的影响

目的筛选灵敏、特异的SARS病毒核酸检测方法。方法采用世界卫生组织(WHO)和中国疾病预防控制中心(中国CDC)推荐的9对SARS病毒核酸检测引物，同时又选择市售的2种SARS荧光定量PCR试剂，分别对不同稀释度的SARS病毒核酸进行RT—PCR、巢式PCR和荧光定量PCR扩增，比较其检测灵敏度和特异性。结果两种荧光定量PCR试剂可检测SARS病毒核酸灵敏度均为0．1TCID50，与普通RT—PCR方法检测灵敏度(0.1TCID50)相同，而较巢式PCR法(0．01TCID50)略低；采用RT—PCR方法扩增SARS核酸时，以WHO推荐的SAR1s／SAR1as、BNIinS／BNIAs、BNIoutS/BnoutAs与中国CDC推荐的CDC-P1 ／P1-灵敏度最高。结论RT—PCR扩增时各引物对之间存在明显差异；荧光定量PCR可作为SARS疑似样本检测的首选方法，必要时再采用RT—PCR和巢式PCR法加以确认。

Influences of different primers on the sensitivity to detect the nucleic acid in SARS virus

To select a sensitive and specific assay for the detection of nucleic acid in SARS virus, the SARS-Cov RNA at different dilutions was amplified by RT-PCR, nested PCR with the 9 pairs primer recommended by WHO and the Center for Disease Control and Prevention of China (CDC). The real-time PCR was performed with two kits for detection of SARS coronavirus. The sensitivity and specificity of these methods were thus compared. It was found that the sensitivity of two kits of real-time PCR for detection of coronavirus was 0.1 TCID_~50 ,that was just the same as that of RT-PCR, but was lower than that of the nested PCR (0.01 TCID_~50 ).When the SARS-Cov RNA was amplified with RT-PCR, the best sensitivities for detection of SARS-Cov RNA were found by using the SAR1s/SAR1as, BN1inS/BN1As, BN1outS/ BN1outAS recommended by WHO and the CDC-P1+/P1-.recommended by CDC. It concludes that there are obvious differences in sensitivity among the 9 pairs of primers with RT-PCR, in which the real-time PCR appears to be the best one that can be used to detect the suspected cases of SARS, and the RT-PCR and nested PCR can be used also if necessary.