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首页> 外文期刊>Journal of Clinical Microbiology >Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses
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Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

机译：登革热和基孔肯雅病毒检测的定量，一步，多重，实时逆转录酶PCR检测方法的开发和验证

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Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping.
机译：登革热病毒（DENV）和基孔肯雅病毒（CHIKV）是重要的人类病原体，具有常见的传播载体和相似的临床表现。在两种病毒共同传播的地区，DENV和CHIKV的误诊可能会影响患者的护理。在这项研究中，我们开发并验证了一步多重逆转录酶PCR（RT-PCR），以同时检测，定量和区分四种DENV血清型（pan-DENV）和基孔肯雅病毒。该测定采用TaqMan技术，采用两个单反应形式的正向引物，三个反向引物和四个荧光团标记的探针。共提取和共扩增的RNA用作内部对照（IC），体外转录的DENV和CHIKV RNA用于生成绝对定量的标准曲线。在线性范围内，诊断（LOD）的95％诊断极限为分别针对DENV（血清型1-4）和CHIKV的50和60 RNA复制/反应。我们的测定法能够检测出53种不同的DENV菌株，分别代表四种血清型和六种CHIKV菌株。没有观察到与相关的黄病毒和α病毒的交叉反应性，为评估诊断的敏感性和特异性，在我们的检测方法中，对89例DENV（1-4型血清型）和CHIKV阳性或阴性的临床样品进行了标准病毒分离方法测试。多重RT-PCR分析显示对DENV的敏感性为95％和100％，对CHIKV的敏感性和特异性为100％。包括RNA提取在内的分析周转时间少于2小时，多重定量RT-PCR分析可为两种临床上难以区分的疾病的差异检测提供快速诊断，这些疾病的地理分布越来越重叠。

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