一步法RT-PCR快速检测鸡新城疫病毒强毒株

Rapid Detection of Virulent Newcastle Disease Virusby One Step Reverse Transcriptase-Polymerase Chain Reaction

本文参照NDV融合蛋白(F)基因序列设计引物,通过一步法RT-PCR扩增到NDV强毒株的170bp大小的特异条带。试验中未检出NDV弱毒株、IBV、AIV、IBDV的RNA,能够检出NDV尿囊液毒的最低滴度为10-5稀释度(相当于103.7EID50病毒量)。对山东省不同地区于1997至2003年所分离的21个NDV强毒株检测全为阳性,而6个NDV弱毒株全为阴性。整个实验操作从病毒核酸提取到判定结果,在5h内即可完成。实验结果表明,该方法特异、敏感。

The one step reverse transcriptase-polymerase chain reaction (RT-PCR) method for detection of virulent Newcastle disease virus (NDV) was developed, and the oligonucleotide primers, selected from the fusion protein-coding gene, amplified the fragments of 170 bp. In these tests, no specific amplification for avirulent NDV, IBV, AIV subtype H5 and IBDV was observed. As little as 10-5-fold dilution of amnio-allantoic fluid containing NDV (approximately 103.7EID50) could be detected with this method. Total of 21 virulent NDV isolates from different areas of Shandong province in 1997～2003 were tested as positive and six avirulent NDV isolates as negative. The process from RNA extraction of virus to result display was completed within five hours. These data suggested that the specific, sensitive and fast method was available for differentiating field case with virulent NDV in chicken.