禽偏肺病毒地高辛核酸探针的制备与应用

Preparation and application of digoxigenin-labeled nucleic acid probe for detection of avian metapneumovirus

根据GenBank中已经发表的B亚型禽偏肺病毒F基因的保守序列设计并合成1对引物,利用RT-PCR扩增出1条与目的片段大小一致的725bp基因片段,回收、纯化PCR产物,用地高辛标记,制备出地高辛标记的aMPV核酸探针。特异性检测结果表明,该探针能与aMPV核酸发生特异性杂交,而与H9N2亚型AIV、NDV、IBV、ORT和E.coil的核酸杂交反应均为阴性;敏感性检测结果表明,该探针对aMPV的最低检出量为5pg。应用制备的探针对山东省不同地区的605份商品肉鸡和122份商品肉鸭进行了核酸探针检测,阳性检出率分别为36.59%和34.51%。本试验制备的aMPV地高辛探针特异性强、敏感性好,对样品的检测结果表明山东省部分地区的商品肉鸡、肉鸭中普遍存在aMPV感染。

According to the genomic sequences of F gene of avian metapneumovirus （aMPV） published in GenBank,a pair of primers was designed for amplifying the 725 hp fragment in RT-PCR experiments. Then the PCR product was labeled with DIG as cDNA probe for detection of aMPV. The hybridization assay of specificity showed that the DNA of aMPV were positive,but other nucleotide extracted from AIV, NDV, IBV, ORT and E. coil were negative. The sensitivity test showed that as low as 5 pg of aMPV＇s DNA could be detected by DIG-labeled probe. The samples of 605 broilers and 122 ducks were collected from flocks in Shandong province,then dot blot hybridization was used to detect avian metapneumovirus,The results show that the detection rates of the two kinds of brids are 36.59% and 34.51% ,respectively. The results showed that the probe could be used in the detection of avian metapneumovirus. This investigation also shows that aMPV was presence in China and the infection is common in broilers and ducks on some farms in Shandong province.