![]() ![]() The column was centrifuged again (110 s, 11,300 RPM) and the flow-through discarded. ![]() The flow-through was discarded and 500 µL of wash 1 was applied to the column. The lysate was applied to a spin column (Omega Bio-Tek Inc., Norcross, USA) and spun in a mySPIN™ 12 (Thermo Fisher Scientific Inc., Waltham, USA) for 110 s at a peak speed of 11,300 RPM. This lysate was hand shaken, then incubated at 61 ☌ for 5 min. Buffers used are described previously by Zainabadi et al. Saliva diluted in UTM (approximately 25% saliva, 75% UTM, 140 µL total) was mixed with 560 µL of a concentrated preparation of lysis buffer and spiked with 2 µL of 50,000 pfu/µL MS2 bacteriophage (Zeptometrix, Buffalo, NY). Instead of adding additional steps to overcome clogging, saliva was collected in UTM. The RNA extraction method was tested with undiluted saliva (neat saliva), however, this resulted in the spin columns getting clogged. This test can be performed on a portable and low-cost device that we manufactured. To meet this immediate need, we developed Saliva-Dry LAMP, a rapid, near-patient saliva test for COVID-19 that uses lyophilized dual-target reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) with fluorometric detection by the naked-eye 11. For remote settings, these tests should not be reliant on cold-chains and sophisticated equipment. Near-patient tests must yield straight-forward results which are easy to interpret. Not surprisingly, governments have pushed for the immediate development of rapid, near-patient tests for COVID-19 9, 10. Tests which require transporting samples to a centralized laboratory increases this time. However, for the sake of contact tracing and self-isolation, the utility of a test relates to how quickly one can receive the results of the test after the sample is obtained 7, 8. This method cannot be deployed outside of a laboratory. Presently, the standard method for COVID-19 diagnostic testing is reverse-transcriptase polymerase chain reaction (RT-PCR) 4– 6. The COVID-19 pandemic has put immense demands on molecular testing infrastructure 2, 3. ![]() The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.ĭue to the highly infectious nature of SARS-CoV-2 and its ability to be transmitted by asymptomatic individuals 1, widespread testing for COVID-19 is critically important to preventing the spread of the virus 1. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. Saliva-Dry LAMP can be completed in 105 min. This test has a limit of detection of 1 copy/µL and achieved a positive percent agreement of 100% and a negative percent agreement of 96.7% relative to a reference standard test. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. The assay relies on dry reagents that are room temperature stable. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye was developed. This study sought to develop a rapid, near-patient saliva-based test for COVID-19 (Saliva-Dry LAMP) with similar accuracy to that of standard RT-PCR tests. This increases the turnaround time for getting test results. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. The highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |