key: cord-337701-56tmg38b
authors: Xiao, Yan; Li, Zhen; Wang, Xinming; Wang, Yingying; Wang, Ying; Wang, Geng; Ren, Lili; Li, Jianguo
title: Comparison of three TaqMan Real-Time Reverse Transcription-PCR assays in detecting SARS-CoV-2
date: 2020-07-06
journal: bioRxiv
DOI: 10.1101/2020.07.06.189860
sha: 
doc_id: 337701
cord_uid: 56tmg38b

Quick and accurate detection of SARS-CoV-2 is critical for COVID-19 control. Dozens of real-time reverse transcription PCR (qRT-PCR) assays have been developed to meet the urgent need of COVID-19 control. However, methodological comparisons among the developed qRT-PCR assays are limited. In the present study, we evaluated the sensitivity, specificity, amplification efficiency, and linear detection ranges of three qRT-PCR assays, including the assays developed by our group (IPBCAMS), and the assays recommended by WHO and China CDC (CCDC). The three qRT-PCR assays exhibited similar sensitivities, with the limit of detection (LOD) at about 10 copies per reaction (except the ORF 1b gene assay in CCDC assays with a LOD at about 100 copies per reaction). No cross reaction with other respiratory viruses were observed in all of the three qRT-PCR assays. Wide linear detection ranges from 106 to 101 copies per reaction and acceptable reproducibility were obtained. By using 25 clinical specimens, the N gene assay of IPBCAMS assays and CCDC assays performed better (with detection rates of 92% and 100%, respectively) than that of the WHO assays (with a detection rate of 60%), and the ORF 1b gene assay in IPBCAMS assays performed better (with a detection rate of 64%) than those of the WHO assays and the CCDC assays (with detection rates of 48% and 20%, respectively). In conclusion, the N gene assays of CCDC assays and IPBCAMS assays and the ORF 1b gene assay of IPBCAMS assays were recommended for qRT-PCR screening of SARS-CoV-2.

(http://www.chinacdc.cn/jkzt/crb/zl/szkb_11803/jszl_11815/202003/W020200309540 91 843062947.pdf) ( Table 1) . Primers and probes were synthesized by standard 92 phosphoramidite chemistry techniques at Qingke biotechnology Co. ltd (Beijing, 93 China). TaqMan probes were labeled with the molecule 6-carboxy-fluroscein (FAM) 94 at the 5' end, and with the quencher Blackhole Quencher 1 (BHQ1) at the 3' end. 95

Optimal concentrations of the primers and probes were determined by cross-titration 96 of serial two-fold dilutions of each primer/probe against a constant amount of purified 97 RNA of SARS-CoV-2. 98

The TaqMan real-time RT-PCR assays were performed by using TaqMan respectively. The concentrations of the RNA transcripts were determined by using 120

NanoDrop (Thermo Fisher Scientific, CA, USA). 121

Comparison of the sensitivities, reproducibility and linear detection ranges of the 123 three qRT-PCR assays. 124

To determine the sensitivity of the three qRT-PCR assays, we measured the limit of 125 detection (LOD) for each assay by using RNA transcript of the corresponding gene in 126 ten-fold dilution as template (RNA transcript alone). A LOD of 10 genomic copies per 127 reaction was observed for both the N gene assay and the ORF 1b gene assay of all the 128 three qRT-PCR assays, although the Ct values for N gene assay of WHO assays and 129 ORF 1b gene assay of CCDC assays were higher than 35 cycles (Table 2) . 130

The linear detection ranges of the three qRT-PCR assays were determined by using a 131 ten-fold dilution of the RNA transcript as template. It showed that the Ct values 132 increased with the RNA transcript from 10 6 to 10 1 copies in the reaction in all of the 133 three qRT-PCR assays ( assays, and CCDC assays, respectively. These results suggested that all of the three 137 qRT-PCR assays exhibited linear detection ranges from 10 6 to 10 1 copies per reaction, 138 while the WHO assays showed lower coefficient of linear correlation. 139

The reproducibility of the three qRT-PCR assays was assessed by measuring 140 coefficient of variation (CV) of mean Ct values in the intra-and inter-assay. For the 141 N gene assay, the CVs of mean Ct values from 10 6 to 10 1 copies of RNA transcript per 142 reaction were 0.20%-1.33%, 0.46%-5.09%, 0.27%-1.97% in intra-assay, and 143 1.06%-2.45%, 0.96%-7.59%, 1.00%-5.51% in inter-assay of IPBCAMS assay, WHO 144 assay, and CCDC assay, respectively. For the ORF 1b gene assay, the CVs of mean Ct 145 values were 0.26%-4.45%, 0.29%-1.76%, 0.71%-6.52% in intra-assay, and 146 2.17%-5.12%, 0.30-1.57%, 2.63%-4.34% in inter-assay of IPBCAMS assays, WHO 147 assays, and CCDC assays, respectively. 148

Because co-infections of respiratory viruses are common, we prepared a (v:v=1:1) 149 mixture of the RNA transcript and a pooled total nucleic acid extract from respiratory 150 specimens (RNA transcript + other extract) as template, to evaluate the effect of 151 co-existed viral nucleic acids on the performance of the assays. No effect of the 152 co-existed other viral nucleic acids on the LOD and the linear detection range was 153 observed, although higher Ct values were generated than those of RNA transcript 154 alone as template in all of the three qRT-PCR assays. However, the co-existed other 155 viral nucleic acids put some effect on the efficiencies of the three qRT-PCR assays. Comparison of the specificities of the three qRT-PCR assays 162

To evaluate the potential cross-reactions with other human respiratory viruses, the 163 three qRT-PCR assays were examined by using human respiratory samples as 164 templates, which were positive for human coronaviruses (OC43, NL63, 229E, or 165 HKU1), or Influenza viruses (A or B), or respiratory syncytial virus, or parainfluenza 166 virus (1-4), or human metapneumovirus, or rhinovirus, or adenovirus, or bocavirus. 167

No cross reaction was observed in all of the three qRT-PCR assays (data not shown), 168

suggesting high specificity of the three qRT-PCR assays in detecting SARS-CoV-2. 169

The three qRT-PCR assays were evaluated with 25 clinical specimens (including 13 171 throat swabs and 12 sputum) from 25 suspected COVID-19 patients. SARS-CoV-2 172 was detected from 92% (23/25), 60% (15/25), 100% (25/25) by the N gene assay, and 173 from 64% (16/25), 48% (12/25), 20% (5/25) of all enrolled clinical specimens by the 174 ORF 1b gene assay in IPBCAMS assays, WHO assays, CCDC assays, respectively 175 (Table 4) . With respect to the sputum, SARS-CoV-2 was detected from 100% (12/12), 176 75% (8/12), 100% (12/12) of specimens by the N gene assay, and from 100% (12/12), 177 75% (8/12), 41.7% (5/12) of specimens by the ORF 1b gene assay in in IPBCAMS 178 assays, WHO assays, CCDC assays, respectively. About the throat swabs, 179 SARS-CoV-2 was detected from 84.6% (11/13), 53.8% (7/13), 100% (12/12) of 180 specimens by the N gene assay, and from 30.8% (4/13), 30.8% (4/13), 0% (0/13) of 181 specimens by the ORF 1b gene assay in in IPBCAMS assays, WHO assays, CCDC 182 assays, respectively. These results demonstrated that the N gene assay performed 183 better than the corresponding ORF 1b gene assay of all the three qRT-PCR assays, the 184 N gene assay in CCDC assays and ORF 1b gene assay in IPBCAMS assays 185 performed better than the other assays. 186

Rapid and accurate detection of SARS-CoV-2 represent a fast-growing global demand, 188 which could be met by TaqMan real time RT-PCR (qRT-PCR). However, the current 189 available TaqMan qRT-PCR assays for SARS-CoV-2 are varied in performance, including sensitivity, specificity, reproducibility, linear detection ranges, etc. Due to 191 that relative lower viral load in upper respiratory tract, reliable qRT-PCR assays for 192 the detection of SARS-CoV-2 are required. We thus compared the performance of 193 three currently wide-applied qRT-PCR assays in the detection of SARS- Sensitivity is the primary demand in the detection of respiratory viruses (6). All of the 195 three qRT-PCR assays could provide a LOD of 10 genomic copies per reaction with a 196 detection range from 10 6 -10 1 genomic copies per reaction. The Ct value at 10 197 genomic copies per reaction in the ORF 1b gene assay of CCDC assays was higher 198 than 35. These results suggested that most of the three qRT-PCR assays provide high 199 sensitivity and wide linear detection range in detecting SARS-CoV-2, except a 200 relative lower sensitivity observed in the ORF 1b gene assay of CCDC assays. 201

Specificity is also essential in the detection of SARS-CoV-2, because of common 202 co-infections with other respiratory viruses and high host DNA background in throat 203 swabs (7-9). We evaluated the specificity of the three qRT-PCR assays with 204 respiratory specimens positive for other common respiratory viruses. No cross 205 reaction was observed, demonstrating high specificity of the three qRT-PCR assays in 206 detection of SARS-CoV-2. 207

We next evaluated the reproducibility of the three qRT-PCR assays by measuring 208 coefficient of variation (CV) of mean Ct values in intra-and inter-assay (10). The N 209 gene assay in IPBCAMS assays and ORF 1b gene assay in WHO assays exhibited a 210 relative better reproducibility with lower intra-and inter-assay CVs, which were not 211 affected by the co-existed nucleic acids of other respiratory viruses. 212

Efficiency is another key parameter of qRT-PCR, reflecting the binding efficiency of 213 primers & probe to template and the amplification efficiency of the PCR system(11). 214

Most of the qRT-PCR assays provided good efficiency, except an abnormal efficiency 215 of 121.83% observed in the ORF 1b gene assay of WHO assays. An exceptionally 216 high efficiency indicates an increased risk of false positive (12). The co-existed 217 nucleic acids of other respiratory viruses increased the efficiency of all the three 218 qRT-PCR assays, suggesting potential increased risk of cross-reactions between the 219 primers & probe and background nucleic acids. 220

We finally evaluate the performance of the three qRT-PCR assays with clinical 221 specimens from suspected SARS-CoV-2 infected patients (13). Possibly because of 222 the lower viral load in upper respiratory tract (4), the detection rate of SARS-CoV-2 223 was lower in throat swabs than in sputum by all of the three assays. Meanwhile, the N gene assay performed better than the corresponding ORF 1b gene assay in all of the 225 three qRT-PCR assays. For the N gene assay, IPBCAMS assays and CCDC assays 226 performed better than WHO assays, both of which could detect SARS-CoV-2 from 227 more than 90% of the suspected specimens. For the ORF1b gene assay, IPBCAMS 228 assays performed better than WHO assays and CCDC assays, with a detection rate of 229 64%. 230

In conclusion, we performed methodological evaluations on three widely-applied 231 qRT-PCR assays for the detection of SARS-CoV-2. Although most of the evaluated 232 assays exhibited good sensitivity, specificity, reproducibility and wide linear detection 233 range, performance test with clinical specimens from suspected COVID-19 patients 234 suggested that the N gene assay in IPBCAMS assays and CCDC assays, and the ORF 235 1b gene assays in IPBCAMS assays were the preferred qRT-PCR assays for accurate 236 detection of SARS-CoV-2. 237 238

The original data will be available upon request. 240 241 RNA extracted from 15 human respiratory specimens by using Trizol. "RNA transcript + other viruses" represents a 1:1 (v/v) mixture of these 269 two components. 

An interactive web-based dashboard to track 275 COVID-19 in real time

Novel coronavirus: From discovery to clinical diagnostics

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The authors declare that there are no conflicts of interest regarding the publication of 243 this paper.