Hoffmann B, Depner K, Schirrmeier H, Beer M
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Boddenblick 5a, D-17493 Greifswald-Insel Riems, Germany.
J Virol Methods. 2006 Sep;136(1-2):200-9. doi: 10.1016/j.jviromet.2006.05.020. Epub 2006 Jun 27.
A heterologous in vitro transcript based on a specific primer-probe HEX system was generated as a universal internal control (IC) to improve virus-specific real-time reverse-transcriptase PCR (RT-PCR) assays. By using a set of different primers, several PCR fragments of desired sizes of an in vitro transcript of the enhanced green fluorescent protein (EGFP) gene were generated, and the fragments were detected using a HEX-labelled probe. For long-term storage of the in vitro transcript a special RNA-safe buffer (RSB) was developed. Freezing and thawing of the IC diluted in RSB did not result in any substantial loss of detectable IC copy numbers. The new IC system was used for the first time in a duplex real-time RT-PCR assay for the detection of pestivirus-derived RNA, in particular from bovine viral diarrhea virus (BVDV). Primers and TaqMan probes for the 'panpesti' assay were selected by analysing the consensus sequence of the 5' non-translated region (5' NTR) of more than 600 different pestiviruses. Finally, the optimised primer probe combination showed an analytical sensitivity of less than 10 copies/reaction. In the duplex set-up, the analytical sensitivity of the validated real-time RT-PCR was identical to the sensitivity of the single assay without IC, and the diagnostic sensitivity of the duplex assay was equal or higher if compared to virus isolation.
基于特定引物-探针HEX系统产生了一种异源体外转录本，作为通用内对照（IC），以改进病毒特异性实时逆转录聚合酶链反应（RT-PCR）检测方法。通过使用一组不同的引物，产生了增强型绿色荧光蛋白（EGFP）基因体外转录本的几个所需大小的PCR片段，并使用HEX标记的探针检测这些片段。为了长期保存体外转录本，开发了一种特殊的RNA安全缓冲液（RSB）。在RSB中稀释的IC冻融不会导致可检测的IC拷贝数有任何实质性损失。新的IC系统首次用于双链实时RT-PCR检测中，以检测瘟病毒衍生的RNA，特别是来自牛病毒性腹泻病毒（BVDV）的RNA。通过分析600多种不同瘟病毒5'非翻译区（5'NTR）的共有序列，选择了用于“泛瘟病毒”检测的引物和TaqMan探针。最后，优化后的引物-探针组合显示出小于10拷贝/反应的分析灵敏度。在双链设置中，经过验证的实时RT-PCR分析灵敏度与无IC的单检测灵敏度相同，并且与病毒分离相比，双链检测的诊断灵敏度相等或更高。
