The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostic platforms. Although several PCR-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid SARS-CoV-2 detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. There are limited reports of saliva as the sample source and some of these indicate inferior sensitivity when comparing RT-LAMP with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay to test saliva using a 2-step RT-LAMP assay, where a short 10-minute RT step is performed with only B3 and BIP primers before the final reaction. We show that while the 1-step RT-LAMP demonstrate satisfactory results, the optimized 2-step approach allows for single molecule sensitivity per reaction and performs significantly better than the 1-step RT-LAMP and conventional 2-step RT-LAMP approaches with all primers included in the RT Step. Importantly, we demonstrate RNA extraction-free RT-LAMP based assays for detection of SARS-CoV-2 from VTM and saliva clinical samples.Competing Interest StatementThe authors have declared no competing interest.Funding StatementThe following reagents were obtained through BEI Resources, NIAID, NIH: (i) Genomic RNA from Zika Virus, PRVABC59, NR-50244; (ii) Zika Virus, PRVABC59, NR-50240. The following reagents were deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: (i) Genomic RNA from SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52285; (ii) SARS-Related Coronavirus 2, Isolate USA-WA1/2020, Heat Inactivated, NR-52286. Authors thank the staff at the Holonyak Micro and Nanotechnology Laboratory at UIUC for facilitating the research and the funding from University of Illinois. R.B. and E.V. acknowledges support of NIH R21 AI146865A, and to also support A.G. A.M. was partially supported by a cooperative agreement with Purdue University and the Agricultural Research Service of the United States Department of Agriculture, AG Sub Purdue 8000074077 to R.B. R.B. and E.V. acknowledge the support of NSF Rapid Response Research (RAPID) grant (Award 2028431). R.B., E.V., A.G. and S.A.S.d.R acknowledge the support of Jump Applied Research through Community Health through Engineering and Simulation (ARCHES) endowment through the Health Care Engineering Systems Center at UIUC. Authors thank Sara Riggenbach, Gabriel Koch, and Bill Bond of OSH Healthcare (Peoria, IL) for their support of the IRB $$ 1602513 and patient sample acquisition for this study. We also thank Mary Ellen Sherwood, Reubin McGuffin, and Carly Skadden of Carle Foundation Hospital (Urbana, IL) for their support of the IRB $$ 20CRU3150 and patient sample acquisition for this study. Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:VTM clinical samples used are discarded viral transport medium prior to the RNA purification step from 25 samples from patients who were tested positive for COVID-19 and from 25 samples from patients who were tested negative for COVID-19 at OSF Healthcare (Peoria, IL) by a RT-PCR test performed at OSF Healthcare. The samples were received de-identified, frozen and were obtained through an approved institutional review board (OSF Peoria IRB $$ 1602513 through the University of Illinois College of Medicine with waiver for consent). Saliva clinical samples were collected from 34 in-patients at Carle Foundation Hospital (Urbana, IL) who were tested COVID-19 positive (RT-PCR) when they were admitted in the hospital (or at most 14 days before admission) through an approved institutional review board (Carle IRB $$ 20CRU3150). Simultaneously to the collection of the saliva clinical samples, nasal swab samples were also collected from the same subject at Carle Foundation Hospital and analyzed by RT-PCR technique at Carle clinical lab. Immediately after collection, the saliva samples were mixed with TE buffer (1:1). Samples were received de-identified and either fresh (during the collection day) or frozen (the next day after the collection day). All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll Data is included in the manuscript.
|Publisher||Cold Spring Harbor Laboratory Press|
- Isothermal amplification
- clinical samples
- RNA extraction-free
- Viral detection