采用二温式聚合酶链反应扩增鸡喉气管炎病毒TK基因的研究

Amplification of Thymidine Kinase Gene of Infectious Laryngotracheitis Virus Using two temperature Polymerase Chain Reaction

建立了检测鸡喉气管炎病毒 (ILTV)的二温式聚合酶链反应 (二温式PCR) ,根据已报道的ILTVTK基因的序列 ,设计并合成了 1对引物 ,用二温式PCR对 5株不同ILTVDNA进行扩增。该对引物对 5株ILTVDNA均扩增出与预期大小相一致的 64 7bp的扩增产物 ,而对新城疫病毒 (NDV )、传染性支气管炎病毒 (IBV)、禽呼肠孤病毒 (ARV)、禽沙门氏菌、鸡毒霉形体和禽巴氏杆菌等 6种禽病病原体的扩增 ,结果全部为阴性。该二温式PCR可检测到 1fg的ILTVDNA模板 ,其敏感性比常规三温式PCR高 1万倍。

A two temperature polymerase chain reaction (PCR) method was developed for detection of infectious laryngotracheitis virus (ILTV). A pair of specific primers were designed and synthesized according to the published sequences of TK gene of ILTV. The DNAs of five strains ILTV were amplificd by two temperature PCR using this pair of primers and ILTV specific 647 base pairs DNA products amplified by this primers were obtained, but not from NDV, IBV, ARV, avian salmonella, fowl mycoplasma and fowl pasteuralla 6 different other avian pathogenic viruses and bacteria. As little as 1 fg ILTV DNA was able to be detected by this two temperature PCR. It is 10 000 times more sensitive than the routine three temperature PCR.