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首页> 外文期刊>Journal of Clinical Microbiology >Development of an Internally Controlled Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Comparison of Four Molecular Dengue Virus Detection Assays
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Development of an Internally Controlled Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Comparison of Four Molecular Dengue Virus Detection Assays

机译：泛登革热病毒检测的内部控制实时逆转录酶PCR检测方法的开发以及四种分子登革热病毒检测方法的比较

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A number of diagnostic tests are available for dengue virus (DENV) detection, including a variety of nucleic acid amplification tests (NAATs). However, reports describing a direct comparison of different NAATs have been limited. In this study, we report the design of an internally controlled real-time reverse transcriptase PCR (rRT-PCR) that detects all four DENV serotypes but does not distinguish between them (the pan-DENV assay). Two hundred clinical samples were then tested using four different DENV RT-PCR assays: the pan-DENV assay, a commercially produced, internally controlled DENV rRT-PCR (the Altona assay), a widely used heminested RT-PCR, and a serotype-specific multiplex rRT-PCR assay. The pan-DENV assay had a linear range extending from 1.0 to 7.0 log10 cDNA equivalents/μl and a lower limit of 95% detection ranging from 1.7 to 7.6 cDNA equivalents/μl, depending on the serotype. When measured against a composite reference standard, the pan-DENV assay proved to be more clinically sensitive than either the Altona or heminested assays, with a sensitivity of 98.0% compared to 72.3% and 78.8%, respectively (P ≤ 0.0001 for both comparisons). The pan-DENV assay detected DENV in significantly more samples collected on or after day 5 of illness and in a subgroup of patients with detectable anti-DENV IgM at presentation. No significant difference in sensitivity was observed between the pan-DENV assay and the multiplex rRT-PCR, despite the presence of an internal control in the former. The detection of DENV RNA late in the course of clinical illness should serve to lengthen the period during which a confirmed molecular diagnosis of DENV infection can be provided.
机译：许多诊断测试可用于登革热病毒（DENV）检测，包括各种核酸扩增测试（NAAT）。但是，描述不同NAAT的直接比较的报告非常有限。在这项研究中，我们报告了一种内部控制的实时逆转录酶PCR（rRT-PCR）的设计，该PCR检测所有四种DENV血清型，但不能区分它们（pan-DENV分析）。然后使用四种不同的DENV RT-PCR测定法对200个临床样品进行了测试：pan-DENV测定法，市售的内部控制的DENV rRT-PCR（Altona测定法），广泛使用的Heminested RT-PCR和血清型特异性多重rRT-PCR分析。 pan-DENV检测的线性范围为1.0至7.0 log 10 cDNA当量/μl，95％检测的下限范围为1.7至7.6 cDNA当量/μl，具体取决于血清型。当使用复合参考标准进行测量时，pan-DENV测定法被证明比Altona或Heminested测定法更具临床敏感性，其灵敏度为98.0％，而分别为72.3％和78.8％（ P ≤0.0001）。在出现疾病的第5天或之后以及在具有可检测的抗DENV IgM的亚组患者中，pan-DENV测定法检测到明显更多的样品中检测到DENV。尽管在前者中存在内部对照，但在pan-DENV测定和多重rRT-PCR之间在灵敏度上没有观察到显着差异。在临床疾病晚期对DENV RNA的检测应有助于延长可以提供DENV感染的分子诊断的期间。

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