Ultrasensitive and fast detection of SARS-CoV-2 using RT-LAMP without pH-dependent dye.

This study investigates the performance of reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the colorimetric detection of SARS-CoV-2 using fluorometric dye, namely, calcein. The detection limit (LoD) with the N-ID1 primer set resulted in superior performance, corresponding to ~ 2 copies/reaction or ~ 0.1 copies/µL of the RNA sample. The color development can be observed by the naked eye, using an ultraviolet (UV) transilluminator or a hand-UV light without the requirement of expensive devices. The average time-to-reaction (TTR) value was 26.2 min in high-copy number samples, while it was about 50 min in rRT-PCR. A mobile application was proposed to quantify the positive and negative results based on the three-color spaces (RGB, Lab, and HSB). Compared to rRT-PCR (n = 67), this assay allows fast and sensitive visual detection of SARS-CoV-2, with high sensitivity (90.9%), selectivity (100%), and accuracy (94.03%). Besides, the assay was sensitive regardless of variants. Since this assay uses a fluorescent dye for visual observation, it can be easily adapted in RT-LAMP assays with high sensitivity. Thus, it can be utilized in low-source centers and field testing such as conferences, sports meetings, refugee camps, companies, and schools.

Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N, RdRP and human RP genes.

Corona Virus Disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pandemic has brought the world to a standstill and threatened human lives. Many methods are known to date to detect this virus. Due to their relative sensitivity, polymerase chain reaction (PCR)-based assays are the most frequently applied and considered the gold standard. However, due to the rapid mutation rate of the viral genome and the emergence of new variants, existing protocols need to be updated and improved. Designing a fast and accurate PCR-based assay is of great importance for the early detection of this virus and more efficient control of the spread of this disease. This study describes a fast, reliable, easy-to-use, and high-throughput multiplex SARS-CoV-2 RT-PCR detection method. The assay was designed to detect two viral genes (N and RdRP) and a human gene (RP) simultaneously. The performance and the sensitivity of the assay were tested in 28 SARS-CoV-2 positive samples and compared with commercial kits, which showed 100% positive percent agreement with a limit of detection (LOD) value of 1.40 and 0.81 copies/µL or 35.13 and 20.31 copies/reaction for RdRP and N genes, respectively. The current assay is found accurate, reliable, simple, sensitive, and specific. It can be used as an optimized SARS-CoV-2 diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

Evaluation of the Idylla KRAS and NRAS mutation test in colorectal cancer tissue.

INTRODUCTION: The currently approved techniques for RAS mutations testing in colorectal cancer (CRC) tissue are labor-intensive and time consuming. The Idylla technology (IT) is a rapid and fully automated diagnostics system. The primary aim of this study is to compare the Idylla performance against that of conventional techniques (CT). METHODOLOGY: Archival CRC tumor samples from 2 hospitals were tested for KRAS and NRAS mutations using the IT. Results were compared to those obtained earlier by CT performed in accredited laboratories. Unexplained discordant results were verified locally by next generation sequencing (NGS) to ascertain the accuracy of IT. RESULTS: Forty five samples were processed. All samples underwent dual testing (CT & IT) for KRAS mutations. IT identified mutations in 2 samples that were not detected by CT. Primary concordance rate for KRAS was 93.3% and the accuracy rate improved to 100% after verification and explanation of discordant results. Only 18 samples underwent dual testing for NRAS. Primary concordance and accuracy rates for NRAS were 94.4%. The mean time from dispatching the specimen for RAS testing by CT until receipt of results was 12 (7-28) days compared to few hours when IT was used. CONCLUSION: IT provides a quick and reliable mean for RAS testing. In addition, it identifies mutations that are not detected by CT and thus may provide better guidance to treatment choices.