key: cord-329395-4k8js9v2
authors: Ratcliff, Jeremy; Nguyen, Dung; Andersson, Monique; Simmonds, Peter
title: Evaluation of Different PCR Assay Formats for Sensitive and Specific Detection of SARS-CoV-2 RNA
date: 2020-07-01
journal: bioRxiv
DOI: 10.1101/2020.06.24.168013
sha: 
doc_id: 329395
cord_uid: 4k8js9v2

Accurate identification of individuals infected with SARS-CoV-2 is crucial for efforts to control the ongoing COVID-19 pandemic. Polymerase chain reaction (PCR)-based assays are the gold standard for detecting viral RNA in patient samples and are used extensively in clinical settings. Most currently used quantitative PCR (RT-qPCRs) rely upon real-time detection of PCR product using specialized laboratory equipment. To enable the application of PCR in resource-poor or non-specialist laboratories, we have developed and evaluated a nested PCR method for SARS-CoV-2 RNA using simple agarose gel electrophoresis for product detection. Using clinical samples tested by conventional qPCR methods and RNA transcripts of defined RNA copy number, the nested PCR based on the RdRP gene demonstrated high sensitivity and specificity for SARS-CoV-2 RNA detection in clinical samples, but showed variable and transcript length-dependent sensitivity for RNA transcripts. Samples and transcripts were further evaluated in an additional N protein real-time quantitative PCR assay. As determined by 50% endpoint detection, the sensitivities of three RT-qPCRs and nested PCR methods varied substantially depending on the transcript target with no method approaching single copy detection. Overall, these findings highlight the need for assay validation and optimization and demonstrate the inability to precisely compare viral quantification from different PCR methodologies without calibration.

SARS-Coronavirus-2 (SARS-CoV-2), a human-infective member of the Betacoronavirus genus 3 (family Coronaviridae), was first identified in the Hubei Province of China in late 2019 as the 4 causative agent behind an increased number of cases of respiratory illness occasionally leading 5 to acute respiratory distress and death. [1] [2] [3] The outbreak was declared a Public Health Emergency 6 of International Concern by the World Health Organization on January 30 th , 2020 and the 7 associated disease was named COVID-19 on February 11 th , 2020. 4, 5 The disease has since 8 spread globally and by June 23 rd has infected nearly 9 million individuals in over 180 countries, 9

causing at least 465,000 deaths. 6,7 10 The ability to accurately identify and diagnose asymptomatically and symptomatically infected 11 patients is crucial for efforts aimed at limiting person-to-person transmission and controlling the 12 outbreak. [8] [9] [10] The standard method for diagnosing viral infections is through the detection of viral 13

nucleic acid in clinical samples. Reverse-transcriptase quantitative polymerase chain reaction 14 (RT-qPCR) is the gold standard used in most diagnostic laboratories. 11 Probe-based RT-qPCR 15 relies on the binding and amplification of three oligonucleotides (two primers and one internal 16 fluorescent probe) and the accumulation of fluorescence signal mediated by DNA polymerase 17 activity. RT-qPCR is not widely accessible as the method relies upon the use of expensive real-18 time PCR platforms and the probe component of the assay is typically the most costly reagent. 19

An alternative, more cost-effective diagnostic method for SARS-CoV-2 RNA is nested PCR. 20

Nested PCR is based on the use of two sequential PCR amplifications wherein the secondary set 21 of primers target sequences nested within the amplicon produced by the first round amplification. 22

Compared to conventional PCR, which uses a single round of replication, nested PCR has 23 increased sensitivity and decreased risk of amplification of non-specific products. 12 Nested PCR 24 methods were developed for SARS, 13 but no nested PCR method for SARS-CoV-2 has yet been 25

In this study, a nested PCR assay for SARS-CoV-2 has been developed and its performance for 

Experiments to determine the sensitivity of the two RT-qPCR methods and nested PCR were 91 completed using serial dilutions of each transcript (5*10 3 to 10 -1 copies/5 µL) in a previously 92 described RNA storage buffer containing RNA storage solution (Thermo Fisher Scientific; 1 mM 93 sodium citrate, pH 6.4), herring sperm carrier RNA (50 µg/mL), and RNasin (New England 94

BioLabs UK, 100 U/mL). 16 For nested PCR experiments, detection rate was assessed over five 95 replicates using the methods described above and a positive result was the presence of a PCR 96 product of the expected length. For RT-qPCR experiments, detection rate was assessed over 97 eight replicate experiments using the methods described above and a positive result was an 98 increase in signal that crossed the threshold value calculated by the machine for each experiment. 99

The CDC N1 method had a cutoff value of Ct 35. The sensitivity of the nested PCR and two RT-qPCR methods was compared by measuring the 118 50% endpoints (50EP) of detection for serial dilutions of the four transcripts described above 119 (Table 1, Figure 2 ). The RdRP transcript does not contain the target sequences of the CDC N1 120 method and thus was not assessed using this method. No method proved to consistently be the 121 most sensitive for all targets, and each method was the most sensitive for at least one target. The between their E, N, and RdRP primer/probe sets, with the RdRP set performing the best with a 156 95% detection probability of 3.8 copies/reaction for SARS-CoV-2 genomic RNA. 14 Corman et al. 157 also reported a 95% detection probability of 5.2 copies/reaction for the E gene assay -this is in 158 conflict with a 95% detection probability of 100 copies/reaction reported by Institut Pasteur using 159 the same primer/probe set. 24 Igloi et al. investigated the performance of 13 commercial RT-qPCR 160 kits and found variable performance between the RT-qPCR kits, with several kits having 10 fold 161 differences in sensitivity for different gene targets and one kit having a 2 log difference in 162 sensitivity between their E and RdRP/N preparations. 25 Using in vitro transcribed small transcripts 163 of five SARS-CoV-2 genes, Vogels et al. evaluated nine primer/probe sets, including the Charité-164

RdRP and CDC N1 sets. 26 Vogels et al. found that eight of the nine primer/probe sets had lower 165 limits of detection of 500 copies/reaction, and that the Charité RdRP set was unable to detect any 166 replicates with 500 copies/reaction, although they did alter the primer and probe concentration This broader experience suggests that the issue may lie within the generalized approach of 175 detecting SARS-CoV-2 RNA by PCR rather than being specific to individual assays. One possible 176 factor limiting the sensitivity of PCR assays for SARS-CoV-2 may originate from the highly 177 structured nature of the genome as measured by standard thermodynamic RNA structure 178 prediction methods. 28 SARS-CoV-2, as well as other coronaviruses, has extensive RNA 179 secondary structure elements peppered throughout its genome -approximately 62% -67% of 180 bases may be involved in base pairing. It is conceivable that, if a section of the target sequence 181 is embedded within highly energetically favoured RNA secondary structure, binding of the PCR 182 oligonucleotides could be competitively inhibited, delaying the initiation of the PCR reaction. This 183 effect could explain the unusual findings that no PCR based assay for SARS-CoV-2, whether in 184 this study or others, have been able to achieve single copy detection sensitivities. The authors report no conflicts of interest. 214 

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Nested PCR detection of clinical samples assessed at the John Radcliffe Hospital via RT-qPCR. COV1-COV36 (panels A-C) were positive samples, NEG1-NEG8 (Panel D) were negative samples. Panel A has a 100 bp molecular ladder

Panels B-D have 1kb molecular ladder

Comparison of detection of SARS-CoV-2 RNA in clinical samples by nested PCR versus qPCR readout from the microbiology unit of the John Radcliffe Hospital. Samples negative via qPCR were given a representative Ct value of 40.