猪瘟RT-PCR ELISA诊断方法的建立

Development of diagnostic RT-PCR ELISA for CSF

将RT-PCR方法和高灵敏度的地高辛检测系统相结合,先建立猪瘟病毒(classical swine fever virus,CSFV)的RT-PCR方法,用地高辛标记RT-PCR产物;再建立CSFV的PCR-ELISA方法,用生物素标记的探针在链亲和素包被的微量板孔中杂交捕获地高辛标记的PCR产物,最后进行免疫显色得出结果。试验主要对RT-PCR ELISA的各种反应条件进行了优化,确定了1mg/L的链亲和素、50μg/L生物素标记的探针、1∶3000抗地高辛的过氧化物酶、37℃杂交2h等为最适反应条件。该方法的敏感性比常规琼脂糖凝胶电泳检测高100倍以上,克服了组织样品中CSFV含量少而难以检测的困难,为猪瘟的早期诊断和分子流行病学调查提供了一条新途径。

In this experiment,the reverse transcription polymerase reaction （RT-PCR） was combined to the high sensitive system of digoxingenin（DIG）. Firstly,the RT-PCR was developed and the RT-PCR product labeled by digoxingenin. Then the RT-PCR ELISA was established to hybridize and capture the DIG-labeled product with the probe which had been binded to the microtiration plate. The results were evaluated by the immunity reaction. The optimal reaction conditions were determined including the probes labelled with 1 mg/L of streptavidin and 50 μg/L of biotine,1 ： 3 000 antidigoxigenin-POD and hybridazation at 37 C for 2 h. This method possess a strong specificity and high sensitivity so that it may overcome the difficulty for the little CSFV in the samples. So it provides a new way for the molecular epidemiologic investigation and early diagonosis of CSFV.