key: cord-1036837-isjg0lr1
authors: Moses, S. E.; Warren, C.; Robinson, P.; Curtis, J.; Asquith, S.; Holme, J.; Jain, N.; Brookes, K. J.; Hanley, Q. S.
title: Endpoint PCR Detection of Sars-CoV-2 RNA
date: 2020-07-29
journal: nan
DOI: 10.1101/2020.07.21.20158337
sha: 0e3ae31e9d67c76c8d3865a3e31d0a8533006d1b
doc_id: 1036837
cord_uid: isjg0lr1

Quantitative real-time PCR methods have been used to perform approximately 278 million tests for COVID-19 up to mid-July 2020. Real-time PCR involves a rate limiting step where the samples are measured in situ during each PCR amplification cycle. This creates a bottleneck limiting scalability and as a consequence reducing access to inexpensive reliable testing at national and international scales. We investigated endpoint PCR for the qualitative detection of SARS-CoV-2 sequences on synthetic RNA standards and hospital patient samples. The endpoint PCR detection limit is constrained only by the stochastics of low copy numbers and reliably detected single copies of synthetic RNA standards. On a set of 30 patient samples, endpoint PCR found one additional positive sample and was able to confirm an indeterminate sample as negative. These results were found using 4 l reagent and 1 l of sample representing an 80% reduction relative to the NHS protocol (20 l reagent and 5 l sample). These results indicate that endpoint PCR should be the method of choice for large scale testing programmes. Based on the experience from ultra-high throughput genotyping efforts a single workflow using 384-well plates has similar PCR capacity (250 Million) to that required for all testing done worldwide during the first 7 month of the pandemic.

Quantitative real-time PCR methods have been used to perform approximately 278 million tests for COVID-19 up to mid-July 2020. Real-time PCR involves a rate limiting step where the samples are measured in situ during each PCR amplification cycle. This creates a bottleneck limiting scalability and as a consequence reducing access to inexpensive reliable testing at national and international scales. We investigated endpoint PCR for the qualitative detection of SARS-CoV-2 sequences on synthetic RNA standards and hospital patient samples. The endpoint PCR detection limit is constrained only by the stochastics of low copy numbers and reliably detected single copies of synthetic RNA standards. On a set of 30 patient samples, endpoint PCR found one additional positive sample and was able to confirm an indeterminate sample as negative. These results were found using 4 μ l reagent and 1 μ l of sample representing an 80% reduction in required RNA extract input and PCR reagent volumes relative to the NHS protocol (20 μ l reagent and 5 μ l sample). These results indicate that endpoint PCR should be the method of choice for large scale testing programmes. Based on the experience from ultra-high throughput genotyping efforts a single workflow using 384well plates has similar PCR capacity (250 Million) to that required for all testing done worldwide during the first 7 month of the pandemic.

COVID-19 has emerged rapidly from a few cases in Wuhan, China, 1-3 to a global pandemic caused by the SARS-CoV-2 virus which is believed to have jumped to humans from an animal host and is one of a host of betacoronaviruses affecting a wide range of animals. [4] [5] [6] [7] This family of pathogens was considered a pandemic threat before the current international crisis began and has defied nearly all attempts to control or eliminate it due to a combination of high infectiousness, undetected carriers and silent transmission. [8] [9] [10] Improved mass scale methods for detecting viral RNA would aid population surveillance for SARS-CoV-2 and put the world in a stronger position for fighting this and other viral diseases.

Real-time PCR is the current standard recommended by the World Health Organisation (WHO) globally for detection of SARS-CoV-2 RNA. This was accepted under emergency regulations in many countries and is now moving to more permanent status. This dominance of testing emerged during the early stages of the pandemic via a WHO listing of diagnostic protocols for COVID-19. 11 At last update, this included seven protocols developed in China, France, USA, Japan, Germany, Hong Kong, and Thailand targeting a range of SARS-CoV-2 sequences. 11, 12 Each protocol used real-time PCR reflecting the availability of instrumentation and expertise in the labs creating the protocols. The universality of real-time PCR methods in the early tests developed for SARS-CoV-2 created a preference in favour of this readout method which has persisted.

Although real-time quantitative PCR is a standard method in diagnostic as well as research labs and universities, 13 it can be argued that real-time fluorescence signal reading and monitoring is not always required for tests that require only a qualitative binary (Yes/No) interpretation for both surveillance as well as diagnostic screening purposes. Industrial scale qualitative PCR using rapid water-bath temperature cycling followed by endpoint readout was . CC-BY-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 July 29, 2020. . https://doi.org/10.1101/2020.07.21.20158337 doi: medRxiv preprint described first in 1993 with a scale of 46,000 parallel reactions in 384-well plates. 14, 15 This progressed to 1536-well formats by 2005 16 and capacity has since grown such that a single lab using endpoint PCR can carry out 1.5 million PCRs per day at reasonable cost for absence/presence SNP genotyping. [17] [18] [19] A single 384-well plate industrial PCR system is capable of 800,000 samples per day (146 M per 6 months) and with full 24/7 could exceed this.

Endpoint PCR and low-cost reagent mixes underpin a cost-saving revolution in genetics such that genotyping is now less costly than phenotyping. [20] [21] [22] As an example, the KASP endpoint assay system underpins hundreds of papers since 2019 (c.f. 19, 22, 23 ). There have been thousands of papers in the last decade proving the robustness of end-point PCR on over 400 genomes particularly for important food crops. Cost and performance comparisons have appeared in the literature and industrial scale PCR cost has been well below US$1/data point for several years and costs as low as US$0.064/data point documented. [22] [23] [24] A similar cost and scale revolution in SARS-CoV-2 testing would enable high-quality monitoring in even the poorest of countries.

Scale limitations create a range of sampling, storage and processing issues. For example, false negatives (FN) have been an ongoing problem with FN rates as high as 50% reported. 25, 26 With limited capacity, retesting of patients or samples is not always feasible. The ultra-high throughput capabilities of endpoint PCR technologies provide an opportunity to correct this.

Further, at least in the UK, the cycle threshold and copy number values provided by real-time PCR are not being used to inform clinical practice in any way and, to our knowledge, this is true worldwide. Therefore, the extra unused information provided by real-time methods is creating an unnecessary and costly bottleneck. Real-time PCR is providing the same Yes/No result for the presence/absence of a short length nucleotide sequence that endpoint PCR can . CC-BY-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 July 29, 2020. . https://doi.org/10.1101/2020.07.21.20158337 doi: medRxiv preprint provide at lower cost and higher throughput. Endpoint PCR has been demonstrated in forensic science 27 and for detection of other infectious diseases such as Hepatitis B, 28 Ebola, 29 and HIV 30 with some authors indicating endpoint methods are more sensitive than real-time. 30 Here, we investigated endpoint PCR as an alternative to real-time PCR for COVID-19

(SARS-CoV-2 RNA) testing. We compare the two techniques on authentic positive samples collected at Kent hospitals during the pandemic and report detection limits using hospital patient samples and a dilution series using synthetic SARS-COV-2 sequences.

Synthetic RNA controls containing nominally 10 6 copies/μl of the Twist Bioscience, Control 2 sequence (GISAID Wuhan-Hu-1; Genbank ID MN908947.3; Twist Bioscience) were diluted to a starting concentration of 100,000 copies per μ l using the mass information provided by the manufacturer. Standards were prepared by serial dilution to obtain 10,000, 1,000, 100, 10, 1, 0.1, and 0.01 copies per μ l in 0.1mM Te (10mM Tris, pH 8.3, 0.1 EDTA).

These were tested using 2 μ l aliquots using 10 replicates.

A set of 30 anonymised combined nasopharyngeal & oropharyngeal samples collected from patients presenting to Kent Hospitals Trust with COVID-19 symptoms were considered.

Using NHS in-house testing 19 of these samples were positive, 10 negative, and 1 indeterminate. All samples were extracted using either the Promega extraction kit or the Abbott M2000 method. The ensuing Promega extracts underwent PCR by either GeneFinder COVID-19 PLUS RealAmp Kit or the Viasure SARS-CoV-2 Real Time PCR Detection Kit.

. CC-BY-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 July 29, 2020. . https://doi.org/10.1101/2020.07.21.20158337 doi: medRxiv preprint

The Abbott M2000 extracts were part of the single-flow closed extraction and PCR analyser process. The specific extraction methods are described below:

Promega Extraction: CC-BY-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 July 29, 2020. 

The endpoint samples and standards were thermally cycled for 50 PCR cycles and quantified using fluorescence readout for TM FAM, TM HEX and TM ROX dyes as needed on a 7900HT (Applied Biosystems) instrument to derive real-time PCR data. The same sample plate was then read with a standard plate reader (Tecan Spark) to generate the endpoint data. The TM ROX signal was used to normalise the acquired data to correct for variations in pipetting across wells. 31

To test the sensitivity of the endpoint system replicate (N = 10) standards for each gene were measured (Figure 1 ) giving 20 measurements per concentration. Of the 160 measurements, one at 200 copies/well resulted in an indeterminate result due to a pipetting failure (no TM ROX was observed). No negative controls amplified. The N1 and N2 replicates gave TM ROX normalised values of 0.0501±0.0017 (N=10) and 0.1052±0.0032 (N = 10), respectively. We considered any value further than 10 sd from the negative controls (0.07 and 0.14) to be detected. Between 20-20,000 copies/well, the genes were detected in all cases except the indeterminate well and formed a tight cluster distant from the negative controls (Figure 1a) .

. CC-BY-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 July 29, 2020. . https://doi.org/10.1101/2020.07.21.20158337 doi: medRxiv preprint Below 20 copies/well, stochastic effects were seen such that the signal levels varied and, as expected, copies were not detected in all wells (Figure 1b, 1c, and 1d) . The 2 copies/well standard amplified sufficiently to be detected in 13 out of 20 cases, 0.2 copies/well amplified in 2 out 20 as did 0.02 copies/well. A simulation consisting of 10,000 repeats of 20 draws from a Poisson distribution having means of 20, 2, 0.2, and 0.02 gave means of 20, 17.2, 3.62, and 0.398 wells, respectively, for the number of wells expected to have at least one copy of the sequences. This was in reasonable agreement the 20, 13, 2, and 2 wells with detected genes. The lowest concentration is high relative to the expected mean; however, our simulation gave 2 or more amplifications in approximately 6% of the 10,000 trials. The variability expected from a Poisson process under these conditions can be seen in the histograms produced by the simulations (Figure 1e ). These results indicate that the endpoint PCR strategy can detect SARS-CoV-2 genes with excellent sensitivity down to levels where variability is dominated by the stochastics of single copies. The quality of the data makes detection of the genes straightforward and well suited to either manual or automated analysis.

. CC-BY-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 July 29, 2020. The standard with 20-20,000 copies per well (a) gave good discrimination between the standard and controls. Below 20 copies/well stochastic effects were seen (panels b, c, and d).

No amplification was observed in the negative controls (black). Panel e shows the results of simulations of a Poisson process corresponding to the expected behavior of 0.02, 0.2, 2, and 20 copies per well.

To test whether the synthetic material gave an unrealistic view of the sensitivity of the method, we evaluated the RNaseP positive and negative controls using the hospital material.

The TM ROX normalised RNaseP measurements gave 1.185±0.036 (N=30) and the negative controls 0.1850±0.0070 (N=6). Any threshold value above 0.255 (10 standard deviations above the negative control) gives very high confidence that a positive sample has been detected in the hospital samples ( Figure 2 ). This yields a simple yes/no answer. Although the positive samples were slightly lower than the positive controls, they form a tight readily interpretable cluster far away from the negative samples and negative controls. This is a . CC-BY-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 July 29, 2020. . https://doi.org/10.1101/2020.07.21.20158337 doi: medRxiv preprint feature of reagent mixes suited to endpoint PCR because they are designed such that the fluorescence intensity reaches the same level independent of the input RNA concentration.

This results in simple interpretation that can be easily automated for large scale screening or done manually. 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 July 29, 2020. . https://doi.org/10.1101/2020.07.21.20158337 doi: medRxiv preprint prominent in the endpoint presentation (Figure 3c ) as the point near the x-axis. This was assigned as a positive. In addition, the indeterminate NHS sample was found to be negative. 

We demonstrated that endpoint PCR is an excellent method for detection of SARS-CoV-2 RNA from patient samples and synthetic standards. Its sensitivity and variability are limited only by the stochastics of low copy numbers. It was able to detect an additional positive sample and resolve an "indeterminate" result as negative. It provides a simplicity of readout giving a clear yes/no answer while removing an equipment bottleneck in testing workflows.

This confirms previous work using endpoint PCR for other diseases. Endpoint PCR is inherently more scalable because the thermocycling step is done in parallel prior to readout . CC-BY-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 July 29, 2020. . https://doi.org/10.1101/2020.07.21.20158337 doi: medRxiv preprint rather than in situ. Real-time PCR limits instrument throughput by monitoring the increased fluorescence as thermocycling takes place. As such, endpoint PCR should be the method of choice for mass testing populations in hospitals, schools, factories, cities, and countries and is readily adaptable to any disease where a sequence is known. Existing real-time PCR labs can switch to endpoint PCR by switching chemistries while using existing equipment which can be replaced as needed. Many countries and industrial organisations may already have much of the required equipment lowering the overall costs and the up-front cost of an installation with 800,000 sample/day capacity is circa £2.3 million. This is insignificant in comparison to the growing loss of life and the economic damage done by the SARS-CoV-2 virus. Even the richest countries will be unable to withstand further extended periods of reduced economic activity leaving an impossible task balancing health and economic hardship. Endpoint PCR methods can provide country scale PCR capacity for . CC-BY-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 July 29, 2020. . https://doi.org/10.1101/2020.07.21.20158337 doi: medRxiv preprint surveillance including multiple retesting and the infrastructure can be adapted to a wide range of diseases as needed.

The data presented and a concordance tables are available as supplementary materials. 

Steve Asquith, Dr. John Holme, and Dr. Nisha Jain are Directors of 3CR Bioscience Ltd, a provider of reagents for PCR including some of the reagents used to produce these results.

They are also persons having significant control of 3CR Bioscience as defined by Companies House where they are listed as company number 10984711.

All other authors declare no competing interests.

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