鸡贫血病病毒套式PCR检测方法的建立及应用

Establishment and Application of an Nested_PCR for the Detection of CAV

鸡贫血病病毒不仅引起鸡的传染性贫血 ,而且也是引起鸡免疫抑制病的主要病原。本研究首次在国内建立了鸡贫血病病毒的套式PCR检测方法。试验证明此方法具有高度的特异性和敏感性 ,以鸡马立克氏病毒、鸡网状内皮组织增生病毒、鸡传染性法氏囊病毒、正常MDCC_MSB1细胞DNA作为模板扩增时均未出现可见带 ,在检测鸡的各种组织病料时均未出现假阳性结果 ,该法比一次性PCR的敏感性高约 1 0 0 0倍 ,能检测到约一个拷贝的鸡贫血病病毒DNA ,而一次性PCR只能检测到3.6× 1 0 3 个拷贝。此方法已成功地应用于临床试验。对于鸡贫血病毒的许多鸡胚分离物和细胞分离物 ,一次性PCR不能扩增出可见电泳条带 ,而套式PCR都能扩增出特异的DNA片段 ,从而说明 ,套式PCR的敏感性大大高于一次性PCR 。

Chicken anemia virus(CAV)not only induces chicken infectious anemia disease,but also is the main pathogen of chicken immunodeficiency disease.A nested_PCR method for the detection of CAV was successfully established.By using the nested PCR specific DNA fragment could be amplified from reference strains,but no perceivable fragment was found when reacted with the control DNAs from Marek's disease virus,Reticulovirus,MDCC_MSBl cells and SPF chicken tissue.It's shown to be highly specific and so sensitive that one copy CAV DNA can be detected.It was successfully applied to the clinical tests,which demonstrated it was more sensitive than the ordinary PCR and able to adjust the pseudo_negative results of the conventional PCR.