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首页> 外文期刊>Frontiers in Microbiology >Endonuclease Restriction-Mediated Real-Time Polymerase Chain Reaction: A Novel Technique for Rapid, Sensitive and Quantitative Detection of Nucleic-Acid Sequence
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Endonuclease Restriction-Mediated Real-Time Polymerase Chain Reaction: A Novel Technique for Rapid, Sensitive and Quantitative Detection of Nucleic-Acid Sequence

机译：核酸内切酶限制介导的实时聚合酶链反应：一种快速，灵敏和定量检测核酸序列的新技术。

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The article reported a novel methodology for real-time PCR analysis of nucleic acids, termed endonuclease restriction-mediated real-time polymerase chain reaction (ET-PCR). Just like PCR, ET-PCR only required one pair of primers. A short sequence, which was recognized by restriction enzyme BstUI, was attached to the 5′ end of the forward (F) or reverse (R) PCR primer, and the new F or R primer was named EF or ER. EF/ER was labeled at the 5′ end with a reporter dye and in the middle with a quenching dye. BstUI cleaves the newly synthesized double-stranded terminal sequences (5′ end recognition sequences and their complementary sequences) during the extension phase, which separates the reporter molecule from the quenching dye, leading to a gain of fluorescence signal. This process is repeated in each amplification cycle and unaffected the exponential synthesis of the PCR amplification. ET-PCR allowed real-time analysis of single or multiple targets in a single vessel, and provided the reproducible quantitation of nucleic acids. The analytical sensitivity and specificity of ET-PCR were successfully evaluated, detecting down to 250 fg of genomic DNA per tube of target pathogen DNA examined, and the positive results were generated in a relatively short period. Moreover, the practical application of ET-PCR for simultaneous detection of multiple target pathogens was also demonstrated in artificially contaminated blood samples. In conclusion, due to the technique’s simplicity of design, reproducible data and low contamination risk, ET-PCR assay is an appealing alternative to conventional approaches currently used for real-time nucleic acid analysis.
机译：该文章报道了一种用于核酸实时PCR分析的新方法，称为核酸内切酶限制介导的实时聚合酶链反应（ET-PCR）。就像PCR一样，ET-PCR只需要一对引物。被限制酶BstUI识别的短序列连接到正向（F）或反向（R）PCR引物的5'末端，新的F或R引物称为EF或ER。 EF / ER在5'末端用报告染料标记，在中间用淬灭染料标记。 BstUI在延伸阶段切割新合成的双链末端序列（5'末端识别序列及其互补序列），从而将报告分子与猝灭染料分离，从而获得荧光信号。在每个扩增循环中重复此过程，并且不影响PCR扩增的指数合成。 ET-PCR可以对单个容器中的单个或多个靶标进行实时分析，并提供可重复的核酸定量。成功评估了ET-PCR的分析灵敏度和特异性，每管检测到的目标病原体DNA可检测低至250 fg的基因组DNA，并在相对较短的时间内产生了阳性结果。此外，在人工污染的血液样本中也证明了ET-PCR在同时检测多种目标病原体中的实际应用。总之，由于该技术的设计简单，可重现的数据和低污染风险，因此ET-PCR测定法是目前用于实时核酸分析的常规方法的一种有吸引力的替代方法。

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