口蹄疫病毒实时RT-PCR检测方法的建立

目的用TaqMan技术建立了一种实时RT-PCR的方法，用于检测口蹄疫病毒，并优选出最佳检测样品。方法设计合成一套引物和TaqMan探针，通过反应条件的优化，建立实时RT-PCR的方法，以扩增口蹄疫病毒的3D基因，并用该法检测患病仔猪的气管、前肢肌肉、皮肤、舌、颈浅淋巴结、脾脏、喉头、食管、腹股沟淋巴结、肺脏、扁桃体、肝脏、肾脏、胸肌、心脏、鼻黏膜、后肢肌肉、脑、骨髓、软腭和血清等样品。结果（1）该方法检测FMDV下限为0．01TCID50其敏感性比普通RT-PCR高100倍，并具有较好的特异性和重复性，（2）FMDV感染仔猪血清、淋巴结、扁桃体和肝脏中病毒含量较高是检测口蹄疫病毒很好的样品。结论建立了一种实时RT-PCR检测口蹄疫病毒的方法。

Establishment of the real-time RT-PCR assay for the detection of foot and mouth disease virus

In the present study, a real-time RT-PCR assay was established on the basis of the TagMan technique for the detection of foot and mouth disease virus (FMDV) in which one set of primers and probe were designed from the nucleotide sequence of 3D gene, and the condition of real-time RT-PCR assay was validated. The assay was evaluated through the detection of the tissue samples, trachea, forelimb muscle, skin, tongue, lymph node, spleen, larynx, esophagus, lung, tonsil, liver , kidney, brisket,heart, mucous membrane of nose, leg muscle, brain, bone marrow, soft palatine and sera from 3 sick piglets. It was found that this assay could be used for the specific and stable detection of FMDV with a limit of detection of 0.01 TCID_ 50 ,which was 100 times higher than that of conventional RT-PCR assay. Higher viral contents of FMDV could be demonstrated in samples of serum, lymph node, tonsil and liver of these 3 sick piglets. It is concluded that a sensitive and rapid method for the detection of FMDV in infected piglets was established successfully.