Rapid and sensitive detection of peste des petits ruminants virus by a polymerase chain reaction assay
Rapid and sensitive detection of peste des petits ruminants virus by a polymerase chain reaction assay
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DOI：
10.1016/s0166-0934(01)00386-x
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发表时间：
2002-02-01
影响因子：
3.1
通讯作者：
Diallo, A
中科院分区：
文献类型：
--
作者：
Couacy-Hymann, E;Roger, F;Diallo, A
建立了一种快速、特异的小反刍兽疫诊断方法。该检测基于玻璃珠上的RNA快速纯化，然后进行逆转录-聚合酶链反应（RT-PCR）。为此，使用一组引物（NP 3/NP 4）来特异性扩增编码小反刍兽疫病毒的核衣壳蛋白的RNA信使的3'末端中约350 bp的片段。PCR产物在琼脂糖凝胶上电泳后通过UV照射检测，或在印迹转移后通过与地高辛-11-dUTP标记的寡核苷酸探针杂交检测。与传统的Vero细胞滴定技术相比，这种RT-PCR检测灵敏度高1000倍。与流行的Chomezynski和Sacchi的方法[Anal. Biochem.162（1987）156]，在玻璃珠上纯化RNA具有更快速且避免使用溶剂的优点。(C)2002 Elsevier Science B. V.保留所有权利。
A rapid and specific test was developed for the diagnosis of peste des petits ruminants disease. This assay is based on the rapid purification of RNA on glass beads followed by the reverse transcription-polymerase chain reaction (RT-PCR). To that effect, a set of primers (NP3/NP4) was used to amplify specifically a fragment of about 350 bp in the 3' end of the RNA messenger that encodes the nucleocapsid protein of the peste des petits ruminants virus. The PCR-product was detected by UV illumination after electrophoresis on agarose gel or by hybridisation with a digoxigenin-11-dUTP labelled oligonucleotide probe after a blot transfer. In comparison with the conventional titration technique on Vero cells, this RT-PCR assay was 1000-fold more sensitive. Compared with the popular Chomezynski and Sacchi's method [Anal. Biochem. 162 (1987) 156], the purification of the RNA on the glass beads offers the advantage of being more rapid and also avoiding the use of solvents. (C) 2002 Elsevier Science B.V. All rights reserved.