Sample Treatment with Trypsin for RT-LAMP COVID-19 Diagnosis

SIMPLE SUMMARY: At the end of 2019, a new virus named SARS-CoV-2, which causes COVID-19 disease, emerged. It was recognized as a pandemic on 11 March 2020 and caused the collapse of hospital systems in several countries due to the overflow of patients. In this context, several detection techniques have been developed, among them, the RT-qPCR achieve the best sensitivity and specificity for virus detection although it is expensive and time-consuming and needs a previous RNA sample extraction. Therefore, the need to speed up the process has led to the development of rapid tests such as antibody-antigen tests, which allow diagnosis in less time, although they have lower sensitivity and specificity. Therefore, we propose a test for SARS-CoV-2 diagnosis that is faster than RT-qPCR and has higher sensitivity and specificity than antigen tests. This test is based on the use of trypsin for sample treatment without an RNA extraction step and the subsequent amplification by Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification. This point-of-care procedure allows COVID-19 diagnosis in 40 min with colorimetric detection in no specialized centers and could be applied to other viruses. ABSTRACT: The SARS-CoV-2 coronavirus is responsible for the COVID-19 pandemic resulting in a global health emergency. Given its rapid spread and high number of infected individuals, a diagnostic tool for a rapid, simple, and cost-effective detection was essential. In this work, we developed a COVID-19 diagnostic test, that incorporates a human internal control, based on the Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP). When working with synthetic SARS-CoV-2 RNA, the optimized RT-LAMP assay has a sensitivity of 10 viral copies and can be detected by fluorescence in less than 15 min or by the naked eye in 25 min using colorimetric RT-LAMP. To avoid the RNA extraction step, a pre-treatment of the sample was optimized. Subsequently, a validation was performed on 268 trypsin treated samples (including nasopharyngeal, buccal, and nasal exudates) and amplified with colorimetric RT-LAMP to evaluate its sensitivity and specificity in comparison with RT-qPCR of extracted samples. The validation results showed a sensitivity and specificity of 100% for samples with Ct ≤ 30. The rapid, simple, and inexpensive RT-LAMP SARS-CoV-2 extraction-free procedure developed may be an alternative test that could be applied for the detection of SARS-CoV-2 or adapted to detect other viruses present in saliva or nasopharyngeal samples with higher sensitivity and specificity of the antibody test.