Development of a rapid LFA test based on direct RT-LAMP for diagnosis of SARS-CoV-2

IF 1.7 Q3 MEDICAL LABORATORY TECHNOLOGY

Practical Laboratory Medicine Pub Date : 2024-11-01 DOI:10.1016/j.plabm.2024.e00437

Negar Sadeghi , Neda Shirazi , Moein Dehbashi , Bahareh Maleki , William C. Cho , Zohreh Hojati

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Abstract

Introduction

In response to the rapid spread of the SARS-CoV-2 virus, we developed a rapid molecular approach to diagnose COVID-19 without the need for RNA extraction.

Methods

The study utilized two molecular methods, RT-qPCR and colorimetric RT-LAMP, to diagnose the RdRp and ORF8 genes, respectively, in oro-nasopharyngeal swabs. Due to the high sequence diversity of ORF8 in SARS-CoV and SARS-CoV-2, it has been identified as a suitable target for virus detection. The RT-LAMP method was also carried out directly on heat-treated swab samples. The strip tests were made using gold nanoparticles and combined with the RT-LAMP for further analysis.

Results

The results showed that the isothermal amplification method had a sensitivity of 95 % (95 % C.I.: 86.08 %–98.96 %) and a specificity of 75 % (95 % C.I.: 19.41 %–99.37 %). The RT-LAMP-LFA method was able to distinguish positive and negative samples with 100 % sensitivity (95 % C.I.: 91.96–100) and 77.27 % specificity (95 % C.I.: 54.63–92.18). This method only required heating swab samples for 10 min at 65 °C before the RT-LAMP reaction.

Conclusion

By utilizing the RT-LAMP in combination with the LFA, it is possible to diagnose SARS-CoV-2 rapidly without the need for RNA extraction. The entire process from sample collection to test interpretation takes only 75–90 min, and the results can be interpreted by untrained individuals with the naked eye. By employing the ORF8 gene as a diagnostic target and eliminating the need for RNA extraction, the direct RT-LAMP-LFA method achieves a significant breakthrough that was not previously reported.

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开发基于直接 RT-LAMP 的快速 LFA 检验，用于诊断 SARS-CoV-2

方法该研究利用 RT-qPCR 和比色 RT-LAMP 两种分子方法分别诊断口鼻咽拭子中的 RdRp 和 ORF8 基因。由于 ORF8 在 SARS-CoV 和 SARS-CoV-2 中具有高度序列多样性，因此被确定为检测病毒的合适目标。RT-LAMP 方法也是直接在经过热处理的咽拭子样本上进行的。结果表明，等温扩增法的灵敏度为 95 %（95 % C.I.：86.08 %-98.96 %），特异性为 75 %（95 % C.I.：19.41 %-99.37 %）。RT-LAMP-LFA 方法能够区分阳性和阴性样本，灵敏度为 100 %（95 % C.I.：91.96-100），特异度为 77.27 %（95 % C.I.：54.63-92.18）。该方法只需在 RT-LAMP 反应前将拭子样本在 65 °C 下加热 10 分钟即可。从样本采集到检验结果判读的整个过程只需 75-90 分钟，未经训练的人也能用肉眼判读结果。直接 RT-LAMP-LFA 方法采用 ORF8 基因作为诊断靶标，无需提取 RNA，实现了前所未有的重大突破。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Practical Laboratory Medicine Health Professions-Radiological and Ultrasound Technology

CiteScore

3.50

自引率

0.00%

发文量

审稿时长

7 weeks

期刊介绍： Practical Laboratory Medicine is a high-quality, peer-reviewed, international open-access journal publishing original research, new methods and critical evaluations, case reports and short papers in the fields of clinical chemistry and laboratory medicine. The objective of the journal is to provide practical information of immediate relevance to workers in clinical laboratories. The primary scope of the journal covers clinical chemistry, hematology, molecular biology and genetics relevant to laboratory medicine, microbiology, immunology, therapeutic drug monitoring and toxicology, laboratory management and informatics. We welcome papers which describe critical evaluations of biomarkers and their role in the diagnosis and treatment of clinically significant disease, validation of commercial and in-house IVD methods, method comparisons, interference reports, the development of new reagents and reference materials, reference range studies and regulatory compliance reports. Manuscripts describing the development of new methods applicable to laboratory medicine (including point-of-care testing) are particularly encouraged, even if preliminary or small scale.