Developing RT-LAMP assays for rapid diagnosis of SARS-CoV-2 in saliva.
Developing RT-LAMP assays for rapid diagnosis of SARS-CoV-2 in saliva.
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DOI：
10.1016/j.ebiom.2021.103736
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发表时间：
2022-01
期刊：
影响因子：
11.1
通讯作者：
Wang X
中科院分区：
文献类型：
--
作者：
Huang X;Tang G;Ismail N;Wang X
由SARS-CoV-2引起的2019冠状病毒病（COVID-19）已导致全球数百万人死亡。当前的危机对快速检测SARS-CoV-2感染产生了前所未有的需求。逆转录环介导等温扩增（RT-LAMP）是一种快速简便的扩增和鉴定目标病原体转录本的方法。然而，与金标准RT-qPCR相比，RT-LAMP的灵敏度和特异性普遍被认为较差。为了解决这一问题，我们将生物信息学和实验分析相结合，以提高新冠肺炎诊断的检测性能。首先，通过实验筛选和高通量测序研究，我们发现了影响LAMP敏感性和特异性的新引物特征。然后利用这些特征构建改进的生物信息学算法来设计针对SARS-CoV-2的LAMP引物。我们进一步严格验证了这些新检测方法的有效性和特异性。我们证明了多重RT-LAMP法可以直接检测唾液中低至1.5拷贝/µL的SARS-CoV-2颗粒，而无需RNA分离。我们使用COVID-19患者的唾液样本进一步测试了这种超敏感和特异性的RT-LAMP检测。临床验证结果表明，新的RT-LAMP检测在总体检测灵敏度和特异性上与标准RT-qPCR相当。综上所述，我们的LAMP引物设计算法和经过验证的检测方法为COVID-19病例的诊断提供了一种快速可靠的方法。国立卫生研究院。
The coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has killed millions of people worldwide. The current crisis has created an unprecedented demand for rapid test of SARS-CoV-2 infection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a fast and convenient method to amplify and identify the transcripts of a targeted pathogen. However, the sensitivity and specificity of RT-LAMP were generally regarded as inferior when compared with the gold standard RT-qPCR. To address this issue, we combined bioinformatic and experimental analyses to improve the assay performance for COVID-19 diagnosis. First, by experimental screening as well as high-throughput sequencing studies, we discovered new primer features that impacted LAMP sensitivity and specificity. These features were then used to build an improved bioinformatics algorithm to design LAMP primers targeting SARS-CoV-2. We further rigorously validated these new assays for their efficacy and specificity. We demonstrated that multiplexed RT-LAMP assay could directly detect as low as 1.5 copies/µL of SARS-CoV-2 particles in saliva, without the need of RNA isolation. We further tested this ultra-sensitive and specific RT-LAMP assay using saliva samples from COVID-19 patients. Clinical validation results indicated that the new RT-LAMP assay was comparable to standard RT-qPCR in overall assay sensitivity and specificity. In summary, our new LAMP primer design algorithm along with the validated assays provide a fast and reliable method for the diagnosis of COVID-19 cases. National Institutes of Health.
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