Rivas-Macho Ane, Sorarrain Ane, Marimón José M, Goñi-de-Cerio Felipe, Olabarria Garbiñe
Gaiker, GAIKER Technology Centre, Basque Research and Technology Alliance, Parque Tecnológico, Ed. 202, 48170 Zamudio, Spain.
Molecular Biology and Biomedicine PhD Program, University of the Basque Country UPV/EHU, 48940 Leioa, Spain.
Diagnostics (Basel). 2023 Jul 11;13(14):2344. doi: 10.3390/diagnostics13142344.
The pandemic situation caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need of fast, simple, and cost-effective tests for the diagnosis of emerging pathogens. RT-qPCR has been established as the reference technique for the diagnosis of SARS-CoV-2 infections. This method requires a time-consuming protocol for the extraction of the nucleic acids present in the sample. A colorimetric reverse transcription loop-mediated isothermal amplification using the calcein molecule combined with a simple extraction-free method for saliva samples (calcein RT-LAMP) has been developed. Samples are heated 95 °C for 10 min before amplification at 63 °C for 40 min. The results can be observed by fluorescence or by the naked eye with a color change from orange to green. The method was compared with commercialized available colorimetric and fluorescent RT-LAMP kits. The developed method shows better sensitivity and specificity than the colorimetric commercial RT-LAMP and the same as the fluorescent RT-LAMP, without the need of a fluorescent reader. Moreover, the calcein RT-LAMP has, compared to RT-qPCR, a sensitivity of 90% and a specificity of 100% for saliva samples with a Ct ≤ 34, without the need for expensive RT-qPCR instruments, demonstrating the potential of this method for population screening.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）引发的大流行形势凸显了对快速、简便且经济高效的新兴病原体诊断检测的需求。逆转录定量聚合酶链反应（RT-qPCR）已成为诊断SARS-CoV-2感染的参考技术。该方法需要耗时的样本核酸提取方案。已开发出一种使用钙黄绿素分子的比色逆转录环介导等温扩增法，并结合一种针对唾液样本的简单免提取方法（钙黄绿素逆转录环介导等温扩增法）。样本在95℃加热10分钟，然后在63℃扩增40分钟。结果可通过荧光观察，也可肉眼观察到颜色从橙色变为绿色。该方法与商业化的比色和荧光逆转录环介导等温扩增试剂盒进行了比较。所开发的方法显示出比商业化比色逆转录环介导等温扩增法更好的灵敏度和特异性，与荧光逆转录环介导等温扩增法相同，且无需荧光读数仪。此外，与RT-qPCR相比，钙黄绿素逆转录环介导等温扩增法对Ct值≤34的唾液样本灵敏度为90%，特异性为100%，无需昂贵的RT-qPCR仪器，证明了该方法在人群筛查中的潜力。
