key: cord-0296302-2hskaaej
authors: Dewhurst, R.; Heinrich, T.; Watt, P.; Ostergaard, P.; Marimon, J. M.; Wood, D.; Koks, S.
title: Validation of a rapid and sensitive SARS-CoV-2 screening system developed for pandemic-scale infection surveillance
date: 2021-11-09
journal: nan
DOI: 10.1101/2021.11.04.21265951
sha: cf51eeebed1e101ac3852a11e5861dc3eb908d49
doc_id: 296302
cord_uid: 2hskaaej

Without any realistic prospect of comprehensive global vaccine coverage and lasting immunity, control of pandemics such as COVID-19 will require implementation of large scale, rapid identification and isolation of infectious individuals to limit further transmission. Here, we describe an automated, high-throughput testing instrument, designed for population-scale testing for SARS-CoV-2 RNA within 25 minutes from inactivated saliva to result, and capable of reporting 3,840 results per hour. This integrated screening platform incorporates continuous flow loading of samples at random intervals to cost-effectively adjust for fluctuations in testing demand. Protecting vulnerable populations during global pandemics requires rapid and sensitive infection surveillance of asymptomatic carriers. This Sentinel surveillance system offers a feasible and scalable approach to complement vaccination, to curb the spread of COVID-19 variants and future pandemics to save lives.

underreporting of prevalence. Based on mortality rates, it was estimated that only 1-2% of COVID-19 cases were initially detected (2) , while underlying SARS-CoV-2 infection being confirmed in only 30% of cases attributed to excess mortality in the US (3, 4) . Clearly, epidemiological viral surveillance measures were not sufficiently effective in halting the rapid spread of infection globally. 5

While there is a wide range in the estimated pre-symptomatic and asymptomatic prevalence of SARS-CoV-2, many SARS-CoV-2-infected persons never go on to develop symptoms (1, 5, 6), or only do so after being infectious for several days. Even before emergence of the delta variant, an extensive systematic review and meta-analysis found that the average asymptomatic 10 prevalence of SARS-CoV-2 infection was 17% (within a reported range of 4-52% (7) ). Since pre-symptomatic and asymptomatic individuals often vary considerably in the load of SARS-CoV-2 virus they carry, surveillance strategies should ideally have sufficient sensitivity to identify all potentially infectious individuals. This will be critical for identifying 'superspreaders', who have been estimated to account for up to 40% of subsequent infections, despite 15 not necessarily having particularly high viral loads in their bodily fluids (including saliva). 'Super-spreaders' are instead postulated to spread the virus more rapidly by alternative means, such as increased aerosol production (8, 9) .

Asymptomatic and pre-symptomatic carriers of SARS-CoV-2.

The combined pre-symptomatic and asymptomatic infection rate has been estimated to exceed 50% in some unvaccinated populations, meaning that testing only symptomatic individuals will fail to detect many SARS-CoV-2 diagnoses. In the UK, 70% of cases were reported as undetected (8) , while a French study found that 93% of infected persons were undetected in a single test (1, 5) . A comprehensive and detailed study from the Vo municipality in Italy, where 25 80% of the population was tested twice for the virus, found that 42% of those infected did not show any symptoms (10) . A more recent report revealed that as much as 59% of all SARS-CoV-2 viral transmission came from asymptomatic transmission events (comprising 35% from presymptomatic individuals and 24% from individuals who never went on to develop COVID-19 symptoms) (11). 30

While vaccination greatly reduces the degree of asymptomatic virus carriage, a recent study showed that viral RNA from SARS-CoV-2 'delta' variant breakthrough infection cases (79% of whom were asymptomatic) could still be detected in vaccinated individuals for up to 33 days (median 21 days) from their original diagnosis (12) . This study found delta viral loads were 251 35 times higher than viral loads reported from infections of previous SARS-CoV-2 strains detected almost a year earlier from the same region using equivalent RT-PCR tests (12) . The increased infectiousness of the delta variant may ultimately be due to these much higher peak viral loads, which were shown to peak within 2-3 days either side of the time of development of symptoms. These findings suggest a more comprehensive vigilance strategy may be required to rapidly 40 detect infectious individuals in the community, irrespective of whether they have symptoms, and regardless or vaccination status, to effectively contain the spread of the more infectious delta variant into vulnerable populations or populations with low prevalence.

Dewhurst et al "Validation of a rapid and sensitive SARS-CoV-2 screening system developed for pandemic-scale infection surveillance"

Responding to these challenges, we focused on developing a population-scale viral surveillance capability by combining sufficiently sensitive chemistry to identify all infectious individuals with a dedicated instrument specifically designed to have the required speed and throughput to support efficient detection and quarantine for effective reduction of transmission. 5 This scalable screening capability is intended to be rapidly deployed at low cost for regular surveillance testing of large numbers of individuals (particularly at borders, ports, and public sporting and entertainment venues). This surveillance system is also intended to enable rapid containment of emerging SARS-CoV-2 variants which might eventually escape vaccine protection. 10

Integrating optimised sample processing & chemistry with an automated screening platform

Here, we report a proof-of-concept pilot for the integration of all the required components of such a testing system, incorporating saliva sample preparation, optimised RT-LAMP (Reverse 15

Transcriptase-Loop Mediated Isothermal Amplification) chemistry and the development of a random access, continuous flow system for scaling the entire process of sample dispensing, incubation, and detection of reactions, while securely reporting results in real time.

We chose to employ RT-LAMP chemistry for rapid virus detection, since its sensitivity is much 20 closer to RT-PCR than to lateral flow Assay (LFA) tests (13, 14) , and because RT-LAMP enzymes are relatively tolerant of inhibitors present in saliva, allowing readout of accurate test results within minutes of direct sample collection (15) .

Identifying robust and sensitive RT-LAMP chemistries for surveillance screening

We first performed a side-by-side comparison of RT-PCR against a range of fluorometric RT-LAMP chemistries utilising five published primer sets and using RNA extracted from 20 clinical saliva samples obtained from individuals who had received a positive nasopharyngeal swab RT-30 PCR test result, including several samples with very low viral loads. For this evaluation we selected the most sensitive RT-PCR assay in the FDA's published list of 117 SARS-CoV-2 tests (Limit of detection (LoD): 180 viral copies/ml) as our benchmark comparator assay (16) . When the same amount of RNA was added to each assay to allow a fair and direct comparison, RT-LAMP detected SARS-CoV-2 virus in 10 out of 20 samples, versus 11 out of 20 for RT- 35 PCR, indicating that RT-LAMP has comparable performance to RT-PCR with the same input RNA amount (Table 1) . We also found good concordance between the time-to-threshold (TTT) values across these RT-LAMP chemistries and cycle threshold (Ct) values in RT-PCR (Suppl. Figure 1 ). The Zhang E1/N2 primer set (17) was the highest performer out of the five primer sets tested in this comparison. This finding led us to include an alternative E1/N2 primer set, 40 which is available commercially for clinical diagnostic use in Europe (Hayat Genetics), in subsequent comparative assays.

Optimizing saliva sample preparation . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted November 9, 2021. ; https://doi.org/10.1101/2021.11.04.21265951 doi: medRxiv preprint Dewhurst et al "Validation of a rapid and sensitive SARS-CoV-2 screening system developed for pandemic-scale infection surveillance"

Large scale surveillance screening applications requiring faster turn-around, may not be compatible with the available testing time window, nor with any equipment required for inclusion of an RNA purification step. A variety of rapid protocols have been developed to address this challenge, such as 'Saliva Direct', which can detect 6-12 copies of SARS-CoV-2 per μl of saliva (18). However, in practice, PCR-based assays have generally proven to be too slow 5 to enable rapid isolation of potentially infectious people who are about to enter or leave crowded high-risk exposure sites such as airplanes. While the literature increasingly supports the use of saliva samples for large-scale surveillance screening (19, 20) , we found saliva testing less straightforward in practice, requiring optimization of collection buffer and heat treatment to ensure robust and sensitive detection using RT-LAMP assays. 10

We therefore optimised sample preparation to allow rapid, sensitive, and repeatable testing directly from saliva without purification of RNA (Direct RT-LAMP). To avoid time-consuming RNA extractions, we trialled a range of different heat inactivation protocols (21, 22) and saliva collection solutions (23, 24), all of which have been found to improve sensitivity in RT-PCR or 15 RT-LAMP assays. However, we adopted a protocol, whereby fresh or frozen saliva is diluted at least 1 in 4 in AviSal Sample Collection Buffer (Hayat Genetics), followed by virus heat inactivation at 95 o C for 10 minutes. This protocol was found to maintain sensitivity of virus detection and allowed for stable storage of the samples at room temperature (see stability data reported below). To make this protocol compatible with our high-throughput surveillance system 20 we refined it by collecting saliva into 96-format tubes pre-filled with AviSal. Sample tubes then transit a fan forced reflow oven for the heat inactivation step. These pre-processed samples are then ready to be analysed within minutes following tube racking and automated uncapping.

Assessing the performance of Direct RT-LAMP for surveillance screening

Using this optimized sample inactivation method, we next compared various dual read-out (fluorometric and colorimetric) direct RT-LAMP chemistries with two approved RT-PCR diagnostic tests, each requiring RNA to be extracted, and with two direct RT-PCR tests, on the same 20 clinical saliva samples ( Table 2) . Direct RT-LAMP detected virus in 15 out of 20 samples, compared to 17 and 18 out of 20 for the two RT-PCR diagnostics assays (Table 2A) . 30 The best RT-LAMP assays could detect virus in all samples corresponding to a Ct of 33/34 and below, where Ct values were derived from an approved diagnostic comparator test (Table 2B) . Encouragingly, detection of samples with low viral loads (Ct >40) was also observed, albeit with less than 100% sensitivity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted November 9, 2021. ; https://doi.org/10.1101/2021.11.04.21265951 doi: medRxiv preprint thought not to be contagious (26) . If we consider only those positive samples with Ct values below 33 (green line in Table 2A ) from potentially infectious individuals, then there is 100% concordance between RT-PCR tests and RT-LAMP targeting the N and E genes ( Table 2) .

Our assay validation studies demonstrated that a variety of RT-LAMP chemistries can be used to 5 detect the SARS-CoV-2 virus directly from as little as 1.25 μl of saliva input diluted in AviSal buffer with high sensitivity, across samples spanning a wide range of viral loads (Ct values ranging from 19 to >40), when compared with RT-PCR "gold standard" comparator assays for detection of SARS-CoV-2 ( Table 2) . Importantly, using the optimised RT-LAMP assay system, we observed 100% sensitivity in detecting SARS-CoV-2 across all specimens tested with 10 potentially contagious viral loads, corresponding to Ct values up to 33. These data suggest that our surveillance system is likely to be sufficiently sensitive to detect all infectious SARS-CoV-2 carriers, given that an individual's viral load beneath our limit of detection are generally thought not to be contagious, given multiple reported failures to cultivate any SARS-CoV-2 virus using in vitro cell culture from samples with Ct values of over minimum reported thresholds ranging 15 from 24 to 33 (26) (27) (28) . Indeed, sequencing of SARS-CoV-2 transcripts from infected cells recently established that full genome sequence coverage was not observed in samples with a Ct greater than 32 (measured with the same Perkin Elmer RT-PCR assay which we used here as our comparator (29), suggesting that any RT-PCR detection of small SARS-CoV-2 genomic fragments in samples beyond this threshold would not likely reflect intact viable virus with 20 potential to be infectious.

Optimising RT-LAMP specificity to minimise false positive reactions

Sequencing of products of LAMP reactions has demonstrated that this amplification chemistry can be prone to the development of false positives in the absence of template, due to non-specific 25 primer-dependent amplification. These non-specific reaction products, the prevalence of which varies between particular primer sets, normally emerge later in the reaction incubation (30), explaining why the 'time-to-positive' relates to specificity as well as to viral load.

Maximum incubation times are therefore typically specified in RT-LAMP protocols to minimise the chance of such false positives arising, even at the expense of reduced sensitivity. We found 30 that in the absence of a sealed heated lid on the Sentinel surveillance instrument, a high rate of evaporation during a standard 30-minute RT-LAMP incubation at 65 o C resulted in the early emergence of false positives after around 20 minutes, causing the reaction specificity to drop below 100%. This finding is particularly relevant to screening on the Sentinel surveillance instrument (see below), which lacks a heated lid seal to minimise evaporation. To better 35 understand the correlation between rate of false positive production and evaporation, we ran multiple negative control colorimetric RT-LAMP reactions in replicate, with both NEB (M1804 plus E1/N2 primers) and Hayat Genetics chemistries using saliva samples negative for SARS-CoV-2 and extended the reaction run time beyond the standard 30 min to 90 minutes (Figure 1 ).

In the absence of mineral oil, 10% of NEB reactions were positive by 30 min, with 100% 40 becoming positive by 40 minutes. The more specific Hayat Genetics assay chemistry was slower to produce false positives, with all reactions still negative at 40 minutes, but eventually turning positive by 70 min, in the absence of mineral oil. These data confirm that RT-LAMP is prone to false positives, and that choice of optimal chemistry and preventing evaporation is critical in ensuring high specificity of RT-LAMP by 30 minutes. The Hayat Genetics assay also exhibited 45 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 9, 2021. ; https://doi.org/10.1101/2021.11.04.21265951 doi: medRxiv preprint superior specificity. However, to further mitigate against the effect of evaporation, regardless of the chemistry used, we incorporated an oil overlay barrier in all RT-LAMP reactions.

We next investigated whether the most effective of the RT-LAMP chemistries we tested (Hayat 5

Genetics) could detect RNA from the dominant "delta" (B.1.617) variant of SARS-CoV-2. As expected, given that the Hayat RT-LAMP primer set amplifies a genomic region which is conserved across SARS-CoV-2 variants, we could robustly detect synthetic RNA from the delta variant across a wide range of concentrations within 20 minutes (Suppl. Figure 2) . 10

Designing a RT-LAMP surveillance system supporting continuous loading of samples at random

We designed an automated RT-LAMP surveillance system, specifically for ultra-high throughput detection of viral nucleic acid, directly from saliva samples taken on a population-wide scale (Figure 2 ). This integrated system combines an optimized system for efficient sample collection 15 preparation in barcoded tubes, which are rapidly heat inactivated and consolidated into racks for automated uncapping and continuous loading onto a robotic instrument which automatically dispenses samples and reagents and continuously scans and reports results.

The integrated system was designed to address key bottlenecks identified in existing surveillance technologies by incorporating the following features: 20 i) A scalable method for safe collection, heat inactivation and tracking of saliva samples.

ii) Optimised sample processing and LAMP assay choice to maximise sensitivity/specificity.

iii) Incorporation of a continuous flow, random access loading of racks of sample 25 tubes/plates onto the system with integrated sample tracking and reporting to support ultra-high throughput applications.

The capacity of conventional molecular diagnostics instruments is constrained by largely batchbased sample loading logistics, frequently resulting in rate-limiting queuing of microplates awaiting access to the instrument. This batch constraint also applies to large 'continuous flow ' 30 diagnostic instruments (31) , which sometimes include multiple incubation stations employed on a range of pre-determined schedules in order to achieve faster cycle times, which can be less time and cost efficient, particularly at lower throughputs, while samples are accumulated to achieve optimal capacity.

To address this scalability bottleneck, the 'Sentinel' LAMP instrument ( Figure 2B ) was designed as a truly continuous flow system, treating the arrival and disposal of each plate into the system completely independently. The Sentinel instrument can perform RT-LAMP tests at up to 3,480 tests per hour, with potential for further increases in scale in the future, by switching from a 96 to a 384 well format. 40

The Sentinel system was conceived to incorporate a resource scheduling and monitoring system tasked with processing as many microplate assays as possible within a given period, balancing upstream processing of samples with downstream reaction and disposal steps, to efficiently . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 9, 2021. ; https://doi.org/10.1101/2021.11.04.21265951 doi: medRxiv preprint Dewhurst et al "Validation of a rapid and sensitive SARS-CoV-2 screening system developed for pandemic-scale infection surveillance" schedule the arrival and departure of microplates being analysed into selected incubation slots within a common isothermal incubation zone -see Suppl. Figure 3 for a video of the Sentinel Instrument.

To establish the feasibility of saliva sample collection without requiring cold chain logistics, we tested how our sample collection procedure could affect the outcome of RT-LAMP assays and challenged the robustness of testing with different storage conditions using AviSal saliva collection buffer. This study established that collected saliva samples, spanning a wide range of viral loads, are stable in this storage media without loss of sensitivity during pre-inactivation 10 storage at RT for up to 48h, post-inactivation at RT for 2h or 4 o C for 48h, and through a freeze/thaw cycle. (Figure 3) .

Colorimetric LAMP limit of detection on the clinically deployed Sentinel instrument 15

Finally, we conducted a study to pilot the implementation of the Sentinel Surveillance instrument in the context of a clinical microbiology service laboratory in San Sebastian, Spain. Saliva samples from individuals who had tested positive by an approved diagnostic RT-PCR swab test were frozen prior to testing on the Sentinel Instrument. Six of these saliva samples, with distinct viral loads, were serially diluted to obtain a panel of 36 samples spanning a wide range of Ct 20 values. These diluted samples were further diluted in a commercially-available saliva transport medium -Saliva Transport buffer M (Vitro SA, Spain) -and heat inactivated for 10 min at 95 o C. Direct RT-PCR (Seegene Allplex) was performed on 5 μl of these heat-treated saliva dilutions. 25 The NEB colorimetric RT-LAMP (N&E-gene, M1800 2x LAMP mix) was run on the Sentinel using 3 μl of the same template as RT-PCR. Higher Ct values could also be detected, but with inconsistent 30 reliability, indicating that the limit of detection for VTM/heat-treated saliva samples combined with NEB M1800/N&E chemistry corresponds to a Ct of 31.1, which represents a slightly less sensitive detection threshold than we obtained for similar chemistry with an alternative AviSal Sample Transport Buffer ( Table 2) . This decreased sensitivity observed may be due to differences in composition of the alternative sample collection buffer used, or alternatively could 35 result from differences in sensitivity between colorimetric/fluorometric chemistries or differences in the comparator RT-PCR assays used in each case. Nevertheless, this pilot implementation study demonstrated that the SARS-CoV-2 RT-LAMP assay is sufficiently versatile to be adapted to be compatible with local sample collection processes of a routine clinical diagnostics service. 40

Addressing the need for more sensitive rapid surveillance of asymptomatic individuals . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 9, 2021. ; https://doi.org/10.1101/2021.11.04.21265951 doi: medRxiv preprint Dewhurst et al "Validation of a rapid and sensitive SARS-CoV-2 screening system developed for pandemic-scale infection surveillance"

While the diagnostic gold standard RT-PCR test is generally sufficiently sensitive to detect all infected individuals, this technology lacks sufficient speed (taking hours) and throughput (at most a few thousand samples processed per instrument per day) to support routine surveillance screening and quarantine applications. In addition, nasopharyngeal sampling requires trained 5 personnel, is inconvenient, and has decreased participant acceptance if multiple testings within short period are required.

Alternative, more acceptable rapid tests are emerging in response to these challenges but are not all sufficiently sensitive to reliably prevent outbreaks, particularly as more infectious variants 10 continue to arise. A recent systematic review suggested an average analytical sensitivity overall for Lateral Flow Antigen (LFA) tests of 72% (32-34), while a systematic review of 58 LFA test evaluations found that their sensitivity was reliable only when detecting samples with high (i.e. Ct <25) viral loads. This explains why their sensitivity was significantly lower in asymptomatic people, where LFA sensitivity ranged from 28% to 69% (32-34). Similarly, a large study of 15 2,215 people attending a diagnostic centre showed the sensitivity of the Roche and Abbott LFA tests to be only 60.4% and 56.8%, respectively, in detecting RT-PCR-positive (Ct<30) individuals (35) , which could still be infectious as outlined above. Real world experience has established that a significant proportion of international air travellers who test positive for SARS-CoV-2 upon arrival are asymptomatic (36, 37) and would therefore would not be reliably 20 detected by lateral flow tests.

Therefore, surveillance testing strategies based solely on such insufficiently sensitive LFA tests may be counterproductive by providing a false sense of security. This was highlighted in an outbreak in Liverpool UK, where 60% of SARS-CoV-2 infections (33% of which had high viral 25 loads) were not detected using LFA (38) ; a Birmingham University study in April 2021 found that only 5% of SARS-CoV-2 -infected individuals were detected by LFA with potentially infectious (Ct<33) viral loads. The recent experience of an outbreak at the Tokyo Olympics, traced to an individual who had tested negative in a LFA test (39) has further highlighted the risks of using LFA tests for asymptomatic surveillance of vulnerable groups. 30

Rapid SARS-CoV-2 Nucleic Acid Test (NAT) technology, such as the Abbott ID Now diagnostic test, has therefore been advocated as an alternative or adjunct to rapid antigen tests for COVID-19 surveillance applications, particularly in the next phase of the pandemic (40) . Yet, a systematic review of pooled data from the multiple clinical evaluations of the Abbott ID Now 35 test found that its sensitivity was inferior to comparator RT-LAMP tests on crude samples (41) .

We have prototyped a RT-LAMP -based screening system, which combines sufficient sensitivity for effective viral surveillance with feasible scalability to very high-throughput. 40 However, given that the viral load in samples from infected but pre-symptomatic or asymptomatic people can take several days to reach the limit of detection of even the most sensitive tests (such as RT-PCR and RT-LAMP), frequent testing may be still be required to maintain effective viral surveillance (42) , especially given evidence of SARS-CoV-2 transmission from asymptomatic people with low viral loads which can gradually increase over 45 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 9, 2021. ; https://doi.org/10.1101/2021.11.04.21265951 doi: medRxiv preprint several days (43) , even in vaccinated people (12) , suggesting that optimal surveillance testing should therefore involve sequential NAT tests.

For example, weekly surveillance testing using saliva-based RT-LAMP has recently been advocated as a feasible means of complementing vaccines, in order to contain the spread of 5 highly infectious pandemic agents such as the SARS-CoV-2 delta variant (44). Travel biosecurity could therefore be enhanced by sequential testing of passengers both before departure, as well as upon arrival, using rapid NAT tests such as RT-LAMP.

The integrated high throughput surveillance system described here allows rapid turnaround from convenient saliva sampling, which may help facilitate community acceptance of repeated testing. 10 Importantly, the confidentiality of data from tested individuals is also maintained within the Sentinel sample tracking system, based on anonymous digital tokens, ensuring that the instrument's reporting of test results remains deidentified. Test results reported from the instrument can only be associated with participant identities externally by authorised entities, such as participants themselves or accredited pathology services, who have exclusive access to 15 the required information.

One limitation of our study was the small number of clinical samples we could readily access for the comparative study, since Western Australia had no community transmission of SARS-CoV-2 between April 2020 and October 2021. Therefore, we had limited opportunity to test our system in the field locally. However, large scale deployments of this system internationally would allow 20 further optimization of testing procedures, adapted to specific feedback from different testing environments. Differences in the operations of seaports, airports and sporting venues or ships, will therefore likely necessitate refinement, to maximise the process efficiency and acceptability.

In conclusion, we have demonstrated the initial proof-of-concept for an optimised saliva-based 25 RT-LAMP assay, integrated with a purpose-built high-throughput viral surveillance instrument. This rapid SARS-CoV-2 vigilance system offers a unique combination of accuracy and scalability to provide a feasible way to mitigate risk of viral transmission as borders open and new viral threats arise in the future.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 9, 2021. ; https://doi.org/10.1101/2021.11.04.21265951 doi: medRxiv preprint Dewhurst et al "Validation of a rapid and sensitive SARS-CoV-2 screening system developed for pandemic-scale infection surveillance"

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 9, 2021. ; Science, Energy and Resources, Australian Government. SK was supported by the Perron Institute for Neurological and Translational Science, Perth, WA. 

Figs. S1 to S2 20 Movies S1

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The copyright holder for this preprint this version posted November 9, 2021. ; https://doi.org/10.1101/2021.11.04.21265951 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 9, 2021. ; https://doi.org/10.1101/2021.11.04.21265951 doi: medRxiv preprint Cycle threshold

Fresh, t=0 + 4 ℃ for 2h

Fresh, t=0 + RT for 2h

Fresh, t=0 + 4 ℃ for 48h

Frozen, t=0 + 4 ℃ for 2h

Frozen, t=0 + RT for 2h

Frozen, RT/6h + 4 ℃ for 2h

Frozen, RT/6h + RT for 2h

Frozen, RT/24h + 4 ℃ for 2h

Frozen, RT/24h + RT for 2h

Frozen, RT/48h + 4 ℃ for 2h

Frozen, RT/48h + RT for 2h

Frozen, 4 ℃/48h + 4 ℃ for 2h 

After heatinactivation . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Ct values; RT-qPCR (PE kit); 2 μl RNA TTT (min); RT-LAMP (NEB E1700 + Syto9); 2 μl RNA RT-qPCR on extracted RNA RT-LAMP on extracted RNA . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted November 9, 2021. ; https://doi.org/10.1101/2021.11.04.21265951 doi: medRxiv preprint Table 3 . RT-LAMP limit of detection in saliva samples carried out on Sentinel instrument in a clinical pathology laboratory. Six SARS-CoV-2 saliva samples with different viral loads were serially diluted 1:10 in nuclease-free water down to 1:100,000 dilutions to obtain 36 samples with a wide range of Ct values. The diluted samples were further diluted 1:3 in Vitro Diagnostica Saliva Transport buffer M (VTM) and heat inactivated for 10 min at 95 o C. Direct 5

RT-PCR (Seegene Allplex) was carried out with 5 ul of heat-treated saliva dilution in VTM; NEB colorimetric RT-LAMP (N&E-gene, M1800 2x LAMP mix) was carried out on a Sentinel station with 3 μl of the same template as RT-PCR. Table A shows the results of RT-PCR and RT-LAMP for all 36 saliva samples organized by sample. Table B shows the same data organized by Ct value. 

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