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以带有鸢尾黄斑病毒的烟草叶片为材料,研究建立了IYSV的普通RT-PCR、免疫捕获RT-PCR和SYBR GreenⅠ实时荧光RT-PCR方法,并比较了几种检测方法的灵敏度。结果表明,DAS-ELISA检测灵敏度较低,为1 mg带毒叶片。而各种PCR方法的灵敏度均高于DAS-ELISA 100倍以上,其中SYBR GreenⅠ实时荧光RT-PCR检测灵敏度最高,可从0.4μg的带毒叶片中检出IYSV,而RT-PCR的灵敏度为40μg带毒叶片,IC-RT-PCR的检测灵敏度是RT-PCR的4倍。鉴于DAS-ELISA灵敏度较低,建议在用ELISA初筛时,如样品OD405值与阴性对照OD405值之比在2.0左右时需要再用分子方法加以确证,以防漏检。
In this study, IYSV RT-PCR, immunocapture RT-PCR and SYBR GreenⅠ real-time fluorescence RT-PCR methods were used to study the sensitivity of several detection methods to tobacco leaves with Iris yellow spot virus. The results showed that the detection sensitivity of DAS-ELISA was low, which was 1 mg infected leaf. The sensitivity of each PCR method was 100 times higher than that of DAS-ELISA. SYBR Green Ⅰ real-time fluorescence RT-PCR assay showed the highest sensitivity, and IYSV was detected from 0.4μg infected leaves. The sensitivity of RT-PCR was 40μg With poisoned leaves, IC-RT-PCR detection sensitivity is four times that of RT-PCR. In view of the lower sensitivity of DAS-ELISA, it is suggested that when screening by ELISA, if the ratio of sample OD405 to negative control OD405 value is around 2.0, molecular method should be used to confirm the result to avoid missing detection.