牛支原体、牛病毒性腹泻病毒和牛传染性鼻气管炎病毒三重二温式PCR检测方法的建立及应用

Development and Application of Two-temperature Triplex PCR for Detecting MB,BVDV and IBRV

为建立一种可同时检测牛支原体(MB)、牛病毒性腹泻病毒(BVDV)和牛传染性支气管炎病毒(IBRV)的三重二温式PCR检测方法。根据GenBank中MB、BVDV和IBRV的保守基因Uvrc基因、5′端非编码区(5′-UTR)和GB基因,分别设计了3对特异引物,将三温式PCR扩增程序简化为两个温度梯度,优化反应条件建立了用于同时检测MB、BVDV以及IBRV 3种常见牛呼吸道传染病病原的三重二温式PCR。结果显示,该方法特异性好,只对MB、BVDV和IBRV模板进行扩增,扩增的目的片段长度分别为412、170、727bp,对其他牛病原体无特异性扩增;灵敏度高,最低能同时检测到10 000拷贝的目的核酸;干扰性小,能同时检测3个不同浓度的模板。研究所建立的MB、BVDV和IBRV三重二温式PCR检测方法,具有特异性好、灵敏度高、方便快速等优点,可用于临床鉴别诊断和流行病学调查,具有很高的临床应用价值。

The aim of this assay was to establish a two-temperature triplex PCR for simultaneously detectingMB,BVDV and IBRV. Three pairs of specific primers were designed and synthesized according to the con-served gene sequences of MB Uvrc,BVDV 5r -UTR and IBRV GB, and a new modified two-temperaturetriplex PCR was developed from tree-temperature conventional PCR for rapid detection of MB, BVDV andIBRV. It was found that the specificity of this assay was high, and able to detect MB, BVDV and IBRVwithout any cross-reactions with other bovine pathogens. The detection limit of the two-temperature triplexPCR assay was 10 000 copies//~L when all of 3 premixed RNA/DNA containing target genes of 3 bovinepathogens were present,indicating a good sensitivity of the assay. Different concentrations of three patho-gens could be identified when mixed together without any interference. All the results indicated that thistwo-temperature triplex PCR assay is specific, sensitive, rapid and simple for the detection of 3 bovine infec-tious diseases. It is a effective tool applied to differential diagnosis rapidly for clinical samples and epidemi-ological investigation.