ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens

Background: Real-Time PCR (RT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. However, due to the low viral load in patient throat and the limitation of RT-PCR, significant numbers of false negative reports are inevitable, which should not be ignored. Methods: We explored the feasibility of droplet digital PCR (ddPCR) to detect SARS-CoV-2 from 57 clinical pharyngeal swab samples and compared with RT-PCR in terms of the sensitivity and accuracy. Among 57 samples, all of which were reported as negative nucleic acid by officially approved clinical RT-PCR detection, 43 samples were collected from suspected patients with fever in clinic, and 14 were from supposed convalescents who were about to discharge after treatment. The experiment was double-blind. Results: The lower limit of detection of the optimized ddPCR is at least 500 times lower than that of RT-PCR. The overall accuracy of ddPCR for clinical detection is 94.3 %. 33 out of 35 negative pharyngeal swab samples checked by RT-PCR were correctly judged by ddPCR based on the follow-up investigation. In addition, 9 out of 14 (64.2 %) supposed convalescents with negative nucleic acid test twice by RT-PCR were positive by ddPCR detection. Conclusions: ddPCR shows superiority for clinical detection of SARS-CoV-2 to reduce the false negatives, which could be a powerful complement to the current standard RT-PCR. Before the ddPCR to be approved for diagnosis, the current clinical practice that the convalescent continues to be quarantined for 2 weeks is reasonable and necessary.


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The recent outbreak of coronavirus disease 2019  caused by the infection 59 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a great 60 threat to public health all over the world. 1,2 On February 28, 2020, the world health 61 organization (WHO) has upgraded the global risk level of this viral pneumonia from 62 "high" to "very high". According to WHO and Chinese Center for Disease Control 63 and Prevention (CDC), the current gold standard for the diagnosis of SARS-CoV-2 64 infection is based on the real-time fluorescent quantitative PCR (RT-PCR), which 65 means that the nucleic acid of SARS-CoV-2 could be detected in patient specimens 66 using 4 However, the disadvantages of insufficient detection of RT-PCR are 67 more and more prominent, especially the problem of detection dynamic range in the 68 clinical application. At present, it has been found in clinical practice that some 69 patients had fever, and chest CT showed symptoms of suspected viral pneumonia such 70 as lower lobe lesions of the lungs, but the nucleic acid test of pharyngeal swab did not 71 show positive results until 5-6 days after the onset of viral pneumonia. It was 72 estimated that only 30 %-60 % positive results can be obtained among  patients that further confirmed by chest CT. 5 This might be explained by the relatively 74 low viral load in the throat of patients and the sensitivity limitation of  technology, which inevitably produced the false negatives during the clinical 76 diagnosis, leading to a potential risk of viral transmission. Besides, supposed 77 convalescent, who is about to discharge, also need multiple tests with negative results 78 for confirmation. Therefore, it is a pressing needs for a more sensitive and accurate 79 detection method for the pathogenic detection. Digital PCR is based on the principles of limited dilution, end-point PCR, and Poisson 82 statistics, with absolute quantification as its heart. 6 It has broader dynamic range 83 without external interference and robustness to variations in PCR efficiency. 7-9 In 84 2011, Hindson developed the droplet digital PCR (ddPCR) technology based on 85 traditional digital PCR. 10 The reaction mixture can be divided into tens of thousands 86 of nanodroplets during the process. These vast and highly consistent oil droplets 87 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https: //doi.org/10.1101//doi.org/10. /2020 substantially improve the detection dynamic range and accuracy of digital PCR in a 88 low-cost and practical format. 11 In recent years, this technology has been widely used, 89 such as analysis of absolute viral load from clinical samples, analysis of gene copy 90 number variation, rare allele detection, gene expression, microRNA analysis and 91 genome edit detection et al. 12,13,14,15 Here, taking the advantages of ddPCR, we 92 optimized the preparation of pharyngeal swab samples, and develop a workflow of 93 ddPCR to detect SARS-CoV-2 using Chinese CDC approved primer and probe sets. 94 Based on the results of this optimized ddPCR system, we showed that the overall 95 accuracy of the ddPCR for clinical pathogen detection is 94.3 %, and 64. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10. 1101/2020 Data statistical analysis 149 Analysis of the ddPCR data was performed with Quanta Soft analysis software

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Comparison of the lower limit between ddPCR and the standard RT-PCR 164 Using a manual threshold to define positivity, 9 % of negative controls (3/32) were 165 scored as positive due to one single positive droplet (data not shown). The presence of 166 two positive droplets or more was not observed for negative controls. Serial dilutions 167 of a positive control DNA fragment of SARS-CoV-2 were tested with primers/probe 168 sets targeting ORF1ab and N of SARS-CoV-2, respectively for ddPCR. It shows good 169 linearity (R2: 0.9932 and 0.9824, respectively) ( Fig. 1A and 1B). Reportable range of 170 ddPCR is from 10 copies/μl to 2500 copies/μl for both ORF1ab and N primes/probe 171 sets. In contrast, the dynamic range of RT-PCR is from 50 copies/μl to 10 5 copies/μl 172 for both ORF1ab and N primes/probe sets ( Fig. 1C and 1D). To define the limit of 173 quantification of ddPCR, five low concentrations of plasmid control were analyzed 174 with 8 replicates. The lower limit of quantitation (LLoQ) of the optimized ddPCR is 175 1.003 copies/μl and 0.415 copies/μl for ORF1ab and N primers/probe sets, 176 respectively. The lower limit of detection (LLoD) of the optimized ddPCR is 0.109 177 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10. 1101/2020 copies/μl and 0.021 copies/μl for ORF1ab and N primers/probe sets, respectively (Fig. 178 2), which is at least 500 times lower than the RT-PCR detection kit used in current 179 clinical test. Therefore, the ddPCR is more sensitive for samples with low level  (Table 1). 199 Secondly, pharyngeal swabs of 8 febrile patients with negative results tested by 200 RT-PCR were also tested negative by ddPCR. In the follow-up investigation 201 COVID-19 was excluded based on the normal results of chest CT and RT-PCR (Table   202 1). 203 Thirdly, pharyngeal swabs collected from 8 febrile suspected patients in the clinic 204 recently with negative nucleic acid tests by RT-PCR, were detected positive by 205 ddPCR. However, chest CT of these 8 patients did not show any abnormalities upon 206 their first visit the clinic. According to official clinical guidelines, these 8 patients 207 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10. 1101/2020 were home quarantined and no further followed-up by us (Table 1). 208 Finally, pharyngeal swabs of 14 supposed convalescent were tested negative in two 209 consecutive tests by RT-PCR (Table 2). However, using ddPCR, 9 out of 14 were 210 positive with a positive rate of 64.2 %. Therefore, the current clinical practice that the 211 convalescent continues to be quarantined for 2 weeks is reasonable and necessary. 212 In conclusion, compared with RT-PCR, ddPCR show superiority for clinical detection 213 of SARS-CoV-2 to reduce the false negatives, which could be a powerful complement 214 to the current standard RT-PCR. precision of RT-PCR for viral quantitation. Different from RT-PCR that the data are 226 measured from a single amplification curve and a Cq value, which is highly 227 dependent on reaction efficiency, primer dimers and sample contaminants, ddPCR is 228 measured at reaction end point which virtually eliminates these potential pitfalls. 229 Results in this work proved that ddPCR is more sensitive (Fig. 1) and accurate for low 230 viral load diagnosis (Fig. 2), which can greatly reduce the false negatives detection 231 (Fig 3). 232 Based on two primers/probe sets targeting ORF1ab and N of SARS-CoV-2, results 233 showed that N primers/probe set was more sensitive compared to that of ORF1ab. 234 Among 42 samples that were judged as positive with ddPCR, 40 in 42 were detected 235 as positive by N primers/probe set, and 12 in 42 were detected as positive by ORF1ab 236 primers/probe set. This could be explained by the subgenomic RNA discontinuous 237 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10. 1101/2020 replication and transcription model of coronavirus. The genome RNA of 238 SARS-CoV-2 encodes single copy of ORF1ab and N, respectively. In contrast, a 239 nested set of around 10 subgenomic RNAs (sgRNAs), each of which encodes one 240 copy of N, are synthesized by viral replication and transcription complex in a manner 241 of discontinuous transcription . 18,19,20 Therefore, the copy numbers of N gene is 242 significantly higher than that of ORF1ab gene in SARS-CoV-2 infected cells. 243 Although 2 patients, who were clinically confirmed by chest CT and RT-PCR 244 subsequently, were reported as negative nucleic acid in pharyngeal swabs by our 245 ddPCR, leading to 2 false negative reports by ddPCR in 35 cases (5.7 % missing rate), 246 the overall accuracy of SARS-CoV-2 detection is significantly improved, which will 247 benefit to the early diagnosis, intervention and treatment. 248 Notably, 64.2 % supposed convalescent patients, who are negative for pharyngeal 249 swab nucleic acid tests twice by RT-PCR, are still carrying SARS-CoV-2 based on our 250 work. Although there is no evidence that such COVID-19 convalescent carrying 251 SARS-CoV-2 will be infectious to other healthy person, the risk still exists. Therefore, 252 the current clinical practice that the convalescent continues to be quarantined for 2 253 weeks is reasonable and necessary. And we recommend that ddPCR could be a 254 complement to the current standard RT-PCR to re-confirm the convalescent, which 255 will benefit to reduce the risk of the SARS-CoV-2 epidemic and social panic. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10. 1101/2020    CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the

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The copyright holder for this preprint  Table 2. Detection results of ddPCR for supposed convalescent patients who is about to be discharged after treatment. .

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