Sample-to-answer, extraction-free, real-time RT-LAMP test for SARS-CoV-2 in nasopharyngeal, nasal, and saliva samples: Implications and use for surveillance testing

Abstract

The global COVID-19 pandemic has highlighted the need for rapid, accurate and accessible nucleic acid tests to enable timely identification of infected individuals. We optimized a sample-to-answer nucleic acid test for SARS-CoV-2 that provides results in <1 hour using inexpensive and readily available reagents. The test workflow includes a simple lysis and viral inactivation protocol followed by direct isothermal amplification of viral RNA using RT-LAMP. The assay was validated using two different instruments, a portable isothermal fluorimeter and a standard thermocycler. Results of the RT-LAMP assay were compared to traditional RT-qPCR for nasopharyngeal swabs, nasal swabs, and saliva collected from a cohort of patients hospitalized due to COVID-19. For all three sample types, positive agreement with RT-LAMP performed using the isothermal fluorimeter was 100% for samples with Ct <30 and 69–91% for samples with Ct <40. Following validation, the test was successfully scaled to test the saliva of up to 400 asymptomatic individuals per day as part of the campus surveillance program at Rice University. Successful development, validation, and scaling of this sample-to-answer, extraction-free real-time RT-LAMP test for SARS-CoV-2 adds a highly adaptable tool to efforts to control the COVID-19 pandemic, and can inform test development strategies for future infectious disease threats.

Development and implementation of a simple and rapid extraction-free saliva SARS-CoV-2 RT-LAMP workflow for workplace surveillance

“…While several saliva-based approaches have been demonstrated for the detection of SARS-CoV-2 using LAMP [ 8 , 14 , 30 , 31 , 35 , 41 – 45 ], including extraction-free or direct protocols [ 13 , 30 , 35 , 41 , 42 , 44 , 46 , 47 ], no gold standard method exists. We focused on the development of a simple and improved sample preparation protocol compatible with this complex sample and downstream LAMP reactions.…”

“…The use of saliva as a less invasive sampling specimen is advantageous since it can be easily self-collected into simple vessels without the need for a healthcare worker, thereby reducing a considerable bottleneck and cost. During the early and acute phases of SARS-CoV-2 infection, a relatively high viral load can be detected in saliva [ 1 – 5 ] and comparative studies show strong agreement in the results obtained from paired nasopharyngeal and saliva samples from the same individual [ 1 , 6 – 13 ]. Importantly, saliva has also been shown to be an effective specimen for the detection of infection in asymptomatic individuals [ 10 , 14 , 15 ] who have a high rate of viral shedding [ 16 ] and therefore pose a significant threat in terms of viral spread.…”

Development and Implementation of a Simple and Rapid Extraction-Free Saliva SARS-CoV-2 RT-LAMP Workflow for Workplace Surveillance

“…While several saliva-based approaches have been demonstrated for the detection of SARS-CoV-2 using LAMP [8,14,30,31,35,[37][38][39][40][41], including extraction-free or direct protocols [13,30,35,37,38,40,42,43], no gold standard method exists. We focused on the development of a simple and improved sample preparation protocol compatible with this complex sample and downstream LAMP reactions.…”

“…The use of saliva as a less invasive sampling specimen is advantageous since it can be easily self-collected into simple vessels without the need for a healthcare worker, thereby reducing a considerable bottleneck and cost. During the early and acute phases of SARS-CoV-2 infection, a relatively high viral load can be detected in saliva [1][2][3][4][5] and comparative studies show strong agreement in the results obtained from paired nasopharyngeal and saliva samples from the same individual [1,[6][7][8][9][10][11][12][13]. Importantly, saliva has also been shown to be an effective specimen for the detection of infection in asymptomatic individuals [10,14,15] who have a high rate of viral shedding [16] and therefore pose a significant threat in terms of viral spread.…”

Scalable RT-LAMP-based SARS-CoV-2 testing for infection surveillance with applications in pandemic control

“…Since the onset of the pandemic, a large number of publications emerged describing the use and validation of RT-LAMP assays for SARS-CoV-2 diagnostics, i.e. (20,27,(38)(39)(40)(41)(42)(43)(44). These publications describe many different primer sets and use patient samples without purification ("direct assays") (some are summarized here: (42)) or with prior purification of RNA, many with optimized procedures where compelling data could be obtained that suggest suitable sensitivity of RT-LAMP for detection of infections in asymptomatic and symptomatic subjects.…”