Stable and noncompetitive RNA internal control for routine clinical diagnostic reverse transcription-PCR
Stable and noncompetitive RNA internal control for routine clinical diagnostic reverse transcription-PCR
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DOI：
10.1128/jcm.42.3.1003-1011.2004
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发表时间：
2004-03-01
影响因子：
9.4
通讯作者：
Jeffery, K
中科院分区：
文献类型：
--
作者：
Dingle, KE;Crook, D;Jeffery, K
基于核酸扩增的临床诊断检测由于速度快、检测限极低，有助于及时诊断微生物感染。然而，为这种测定设计适当的内部控制已被证明是困难的。我们描述了一种用于诊断逆转录(RT)-PCR的反应特异性RNA内部控制，它允许提取，RT，扩增和检测被监测。对照包括一个富含G+ c（60%）的RNA分子，具有广泛的二级结构，基于修饰的丁型肝炎病毒基因组。该RNA的棒状结构具有70%的分子内碱基配对，为RT-PCR提供了一个困难的模板。这确保了更有利的目标病毒扩增子优先于对照产生，只有当目标病毒不存在时才检测到对照。丁型肝炎病毒RNA的不寻常结构先前已被证明可增强其稳定性和对核酸酶的抗性，这是常规使用作为内部控制的优势。对照采用3种巢式多重rt - pcr检测9种临床上重要的呼吸道病毒：(i)甲型流感病毒和乙型流感病毒，（ii）呼吸道合胞病毒A、B和人偏肺病毒，以及（iii）副流感病毒1 ~ 4型。这些试验的检测限不受RNA对照存在的明显影响。在对324个连续的未选择的呼吸样本进行常规检测时，内部控制的存在确保了真阴性和假阴性结果的区分，从而增加了检测的诊断信心。
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.