小鹅瘟PCR诊断方法的建立和初步应用

根据发表的鹅细小病毒B株VP3基因序列 ,设计合成了 1对寡聚核苷酸引物。以国内分离的小鹅瘟病毒为摸板 ,筛选了 (PCR)最佳反应条件 ,并进行了敏感性、特异性和初步应用试验。结果表明 ,在 4 0 μL体积中 ,PCR最佳系统组成为 2uTaq酶、0 5mol/LdNTP和 2 0 pmol引物 ;最佳反应参数为 96℃变性 4 0s ,5 4℃退火 1min ,72℃延伸 1min ,30个循环。本方法可检出 2 5× 10 -1 5EID50 小鹅瘟病毒 ,并且仅能从小鹅瘟病毒的鹅胚培养物中扩增出与设计值大小相同的 375bp核苷酸片断 ,对照病毒扩增结果为阴性。通过对 6份临床病料的检查 ,2份呈阳性 ,其中 1份来自有典型小鹅瘟症状的雏鹅 ,1份来自无典型小鹅瘟症状的雏鹅。由此说明 ,本试验所建立的诊断小鹅瘟的PCR方法具有潜在的临床应用价值

Establishment and Primary Application of PCR for Diagnosis of Gosling Plaque

A PCR method for diagnosing the gosling plaque was established with a pair of primers designed and synthesized based on the VP3 gene of the B strain of goose parvovirus The condition for PCR had been optimized And then the sensitivity, specificity and primary application tests were carried out The optimal PCR system consisted 2u Taq polymerase, 0 5 mol dNTP and 20 pmol primers etc in 40 μL volume and the reaction condition were 96℃ for 40 for sec, 54℃ for 1 min ,72℃ for 1 min and 30 cycles The PCR method could detect 2 5×10 -1 5 EID 50 of the goose parvovirus isolated in China The specificity of the method was very high Using the method, 2 of 6 clinical samples were positive for gosling plaque, one with gosling plaque syndromes, and the other not