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Colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a visual diagnostic platform for the detection of the emerging coronavirus SARS-CoV-2
Analyst ( IF 3.6 ) Pub Date : 2020-10-28 , DOI: 10.1039/d0an01775b
Kawin Nawattanapaiboon 1, 2, 3, 4, 5 , Ekawat Pasomsub 3, 6, 7, 8, 9 , Photchanathorn Prombun 1, 2, 3, 4 , Akanit Wongbunmak 1, 2, 3, 4 , Akarawit Jenjitwanich 1, 2, 3, 4 , Pantanat Mahasupachai 1, 2, 3, 4 , Purichaya Vetcho 1, 2, 3, 4 , Cholticha Chayrach 1, 2, 3, 4 , Natthapon Manatjaroenlap 1, 2, 3, 4 , Chonchanok Samphaongern 1, 2, 3, 4 , Treewat Watthanachockchai 3, 6, 7, 8, 9 , Phonthanat Leedorkmai 3, 6, 7, 8, 9 , Suwimon Manopwisedjaroen 3, 4, 9, 10, 11 , Radeekorn Akkarawongsapat 3, 4, 9, 10, 11 , Arunee Thitithanyanont 3, 4, 9, 10, 11 , Matthew Phanchana 3, 4, 9, 12, 13 , Watanalai Panbangred 3, 4, 9, 11, 14 , Somchai Chauvatcharin 3, 4, 9, 11, 14 , Toemsak Srikhirin 3, 4, 5, 9, 11
Affiliation  

COVID-19, caused by the infection of SARS-CoV-2, has emerged as a rapidly spreading infection. The disease has now reached the level of a global pandemic and as a result a more rapid and simple detection method is imperative to curb the spread of the virus. We aimed to develop a visual diagnostic platform for SARS-CoV-2 based on colorimetric RT-LAMP with levels of sensitivity and specificity comparable to that of commercial qRT-PCR assays. In this work, the primers were designed to target a conserved region of the RNA-dependent RNA polymerase gene (RdRp). The assay was characterized for its sensitivity and specificity, and validated with clinical specimens collected in Thailand. The developed colorimetric RT-LAMP assay could amplify the target gene and enabled visual interpretation in 60 min at 65 °C. No cross-reactivity with six other common human respiratory viruses (influenza A virus subtypes H1 and H3, influenza B virus, respiratory syncytial virus types A and B, and human metapneumovirus) and five other human coronaviruses (MERS-CoV, HKU-1, OC43, 229E and NL63) was observed. The limit of detection was 25 copies per reaction when evaluated with contrived specimens. However, the detection rate at this concentration fell to 95.8% when the incubation time was reduced from 60 to 30 min. The diagnostic performance of the developed RT-LAMP assay was evaluated in 2120 clinical specimens and compared with the commercial qRT-PCR. The results revealed high sensitivity and specificity of 95.74% and 99.95%, respectively. The overall accuracy of the RT-LAMP assay was determined to be 99.86%. In summary, our results indicate that the developed colorimetric RT-LAMP provides a simple, sensitive and reliable approach for the detection of SARS-CoV-2 in clinical samples, implying its beneficial use as a diagnostic platform for COVID-19 screening.

中文翻译:

比色逆转录环介导的等温扩增(RT-LAMP)作为检测新兴冠状病毒SARS-CoV-2的视觉诊断平台

由SARS-CoV-2感染引起的COVID-19已作为快速传播的感染出现。该疾病现已达到全球大流行的程度,因此必须采取更快速,更简单的检测方法来遏制病毒的传播。我们旨在开发基于比色RT-LAMP的SARS-CoV-2视觉诊断平台,其灵敏度和特异性水平可与商业qRT-PCR分析相媲美。在这项工作中,设计引物以靶向RNA依赖性RNA聚合酶基因(RdRp)的保守区域。该测定法以其灵敏度和特异性为特征,并用泰国收集的临床标本进行了验证。所开发的比色RT-LAMP分析可以扩增目标基因,并在65°C下60分钟内进行视觉解释。与其他六种常见人类呼吸道病毒(甲型流感病毒H1和H3,乙型流感病毒,呼吸道合胞病毒A和B型以及人间质肺病毒)和其他五种人类冠状病毒(MERS-CoV,HKU-1,观察到OC43、229E和NL63)。用人造样品评估时,每个反应的检出限为25个拷贝。但是,当孵育时间从60分钟减少到30分钟时,此浓度下的检出率降至95.8%。在2120个临床样本中评估了开发的RT-LAMP分析的诊断性能,并与商业qRT-PCR进行了比较。结果显示高灵敏度和特异性分别为95.74%和99.95%。RT-LAMP测定的总体准确性确定为99.86%。综上所述,
更新日期:2020-11-09
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