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

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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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