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

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.

The Role and Value of Professional Rapid Testing of Acute Respiratory Infections (ARIs) in Europe: A Special Focus on the Czech Republic, Poland, and Romania

“…The implementation of molecular tests can be hampered by the need for the specific training of healthcare providers and adequate infrastructure [ 212 , 213 ], while new rapid options such as NEAR and LAMP (especially automated formats) might be viable alternatives [ 43 , 44 , 45 , 46 , 47 , 50 ]. Multiplex tests might provide a more accurate diagnosis using a single sample and subsequently save valuable time in accessing life-saving treatment in severe or high-risk cases [ 214 , 215 , 216 ], meaning these tests are noteworthy options for hospital settings [ 61 ].…”

“…The reverse transcription LAMP (RT-LAMP) is a viable alternative to RT-qPCR for POC due to its specificity and sensitivity (nears that of RT-qPCR), cost-effectiveness, minimal instrumentation needed (simplicity), and clinically relevant turnaround time [ 43 , 44 , 45 , 46 , 47 ]. However, this technology, like any other, has its limitations, mainly due to its risk of false positives [ 43 , 44 ], due to unintended primer cross-reactivity [ 47 ] or the pH-based colorimetry used in LAMP [ 43 ].…”

“…However, they might be difficult to implement in decentralized healthcare settings (e.g., POC) and emergency rooms due to their general higher cost, longer turnaround time, and the conditions required for their application and processing (the type of equipment needed and sample storage requirements) [ 204 ]. While there are some rapid options available, such as NEAR and LAMP, which can detect infection in a reasonable timeframe (5 to 30 min) and can easily be implemented at POC [ 43 , 44 , 45 , 46 , 47 , 49 , 50 , 52 ], they also carry some limitations associated with intrinsic test characteristics (described earlier). This can lead to a risk of false positives (in the case of LAMP) [ 43 , 44 ] and false negatives (in the case of NEAR) [ 43 , 53 ].…”

Self-Diagnosis of SARS-CoV-2 from Saliva Samples at Home: Isothermal Amplification Enabled by Do-It-Yourself Portable Incubators and Laminated Poly-ethyl Sulfonate Membranes

“…Indeed, general evidence indicates that the inclusion of protease K in extraction-free protocols improves performance, which is generally measured as a decrease in the cycle threshold (CT) in RT-PCR methods or as an improvement in the limits of detection in LAMP-based studies [14,44]. The addition of protease K or trypsin [45] to optimize extraction should be explored in future studies.…”