猪瘟RT-PCR ELISA诊断方法的建立及其初步应用

Development and application of RT-PCR ELISA to diagnosis of classical swine fever virus

建立了检测CSFV的快速、敏感、特异的RT PCRELISA方法。在RT PCR过程中以地高辛标记的dUTP(DIG dUTP)标记PCR产物 ,用生物素标记的捕获探针在链亲和素包被微量板孔中杂交捕获PCR产物。该方法的敏感性较常规RT PCR方法高 10 0倍 ,可检测出 35 0 μL1∶10 0 (相当于 3.5mg)的兔脾组织悬液中的C株CSFV ,特异性强 ,避免了PCR过程中因污染导致的假阳性结果。通过对RT PCR和微量杂交系统的条件优化 ,该方法用于检测C株兔脾组织毒、细胞适应毒和野毒感染的猪组织毒样品 。

A sensitive and rapid detection method for classical swine fever virus (CSFV) was developed. The primers and probe were selected from the 5′ UTR region. The specific amplification products of CSFV RNA by reverse transcription polymerase chain reaction (RT PCR) were labelled with digoxigenin (DIG) during the amplification and the CSFV specific capture probe was labelled with biotin which enables colorimetric assessment of hybridization reactions in microwell plates ELISA. By serial dilutions of DIG labelled PCR products, the RT PCR ELISA was found to be 100 times more sensitive than the conventional agarose gel stained with ethidium brimide(EB) and it can detect the C strain CSFV of 350 μL supernatant of rabbit spleen tissue which were diluted in 100 times (equal to 3.5 mg). The specificity of the method was also very well, it reduced the possibility of contamination during PCR and false positive results.