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首页> 外文期刊>Journal of Clinical Microbiology >Stable and Noncompetitive RNA Internal Control for Routine Clinical Diagnostic Reverse Transcription-PCR
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Stable and Noncompetitive RNA Internal Control for Routine Clinical Diagnostic Reverse Transcription-PCR

机译：用于常规临床诊断逆转录PCR的稳定和非竞争性RNA内部控制

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Clinical diagnostic tests based on nucleic acid amplification assist with the prompt diagnosis of microbial infections because of their speeds and extremely low limits of detection. However, the design of appropriate internal controls for such assays has proven difficult. We describe a reaction-specific RNA internal control for diagnostic reverse transcription (RT)-PCR which allows extraction, RT, amplification, and detection to be monitored. The control consists of a G+C-rich (60%) RNA molecule with an extensive secondary structure, based on a modified hepatitis delta virus genome. The rod-like structure of this RNA, with 70% intramolecular base pairing, provides a difficult template for RT-PCR. This ensures that the more favorable target virus amplicon is generated in preference to the control, with the control being detected only if the target virus is absent. The unusual structure of hepatitis delta virus RNA has previously been shown to enhance its stability and resistance to nucleases, an advantage for routine use as an internal control. The control was implemented in three nested multiplex RT-PCRs to detect nine clinically important respiratory viruses: (i) influenza A and B viruses, (ii) respiratory syncytial viruses A and B and human metapneumovirus, and (iii) parainfluenza virus types 1 to 4. The detection limits of these assays were not detectably compromised by the presence of the RNA control. During routine testing of 324 consecutive unselected respiratory samples, the presence of the internal control ensured that genuine and false-negative results were distinguishable, thus increasing the diagnostic confidence in the assay.
机译：基于核酸扩增的临床诊断测试因其速度快，检出限极低而有助于迅速诊断微生物感染。但是，已经证明设计用于这种测定的适当内部对照是困难的。我们描述了一种用于诊断性逆转录（RT）-PCR的反应特异性RNA内部对照，它可以监控提取，RT，扩增和检测。对照物由富含G + C的（60％）RNA分子组成，该分子具有广泛的二级结构，基于改良的肝炎三角洲病毒基因组。这种RNA的棒状结构具有70％的分子内碱基配对，为RT-PCR提供了困难的模板。这确保了优先于对照产生更有利的靶病毒扩增子，仅当不存在靶病毒时才检测到对照。先前已证明，肝炎三角洲病毒RNA的异常结构可增强其稳定性和对核酸酶的抵抗力，这是常规用作内部对照的优势。对照在三个嵌套的多重RT-PCR中实施，以检测9种临床上重要的呼吸道病毒：（i）甲型和乙型流感病毒，（ii）呼吸道合胞病毒A和B和人间质肺病毒，以及（iii）1型至5型副流感病毒4.这些检测的检出限不会因RNA对照的存在而受到可检测的损害。在对324个连续未选择的呼吸道样本进行常规测试的过程中，内部对照的存在确保了真实和假阴性结果是可区分的，从而增加了对测定方法的诊断信心。

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