key: cord-0707604-czz4jtqp
authors: Ishige, Takayuki; Murata, Shota; Taniguchi, Toshibumi; Miyabe, Akiko; Kitamura, Kouichi; Kawasaki, Kenji; Nishimura, Motoi; Igari, Hidetoshi; Matsushita, Kazuyuki
title: Highly sensitive detection of SARS-CoV-2 RNA by multiplex rRT-PCR for molecular diagnosis of COVID-19 by clinical laboratories
date: 2020-04-23
journal: Clin Chim Acta
DOI: 10.1016/j.cca.2020.04.023
sha: cc11bbcca30ca2002b32b857b034a02f5a330903
doc_id: 707604
cord_uid: czz4jtqp

Abstract Background The detection of SARS-CoV-2 RNA by real-time reverse transcription–polymerase chain reaction (rRT-PCR) is used to confirm the clinical diagnosis of COVID-19 by molecular diagnostic laboratories. We developed a multiplex rRT-PCR methodology for the detection of SARS-CoV-2 RNA. Methods Three genes were used for multiplex rRT-PCR: the Sarbecovirus specific E gene, the SARS-CoV-2 specific N gene, and the human ABL1 gene as an internal control. Results Good correlation of Cq values was observed between the simplex and multiplex rRT-PCR methodologies. Low copies (<25 copies/reaction) of SARS-CoV-2 RNA were detected by the novel multiplex rRT-PCR method. Conclusion The proposed multiplex rRT-PCR methodology will enable highly sensitive detection of SARS-CoV-2 RNA, reducing reagent use and cost, and time required by clinical laboratory technicians.

The global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute 4 respiratory syndrome coronavirus 2 (SARS-CoV-2) was declared by the World Health 5 Organization (WHO) in March 2020 [1] . Currently, clinical diagnosis of COVID-19 is 6 confirmed by detecting SARS-CoV-2 RNA by molecular diagnostic laboratories using the real-7 time reverse transcription-polymerase chain reaction (rRT-PCR) [2] [3] [4] [5] . 8 We analyzed two regions of the N genes used to detect SARS-CoV-2 RNA by simplex one-9 step rRT-PCR using the previously described primer/probe sets N_Sarbeco and NIID_2019- 29 Three genes were designed: a Sarbecovirus-specific E gene (E_Sarbeco, target), a SARS-CoV- 37 We used a synthetic RNA control with the N gene (CTRL-N) with a previously described 

The LOD was analyzed using serially diluted synthetic control RNA samples (2500, 250, 25, 87 5 and 2.5 copies/reaction). The C q values and detection rates are shown in Table 1 93 We compared the three primer/probe sets NIID-N, N_Sarbeco and E_Sarbeco. The E_Sarbeco 102 We compared the C q values of simplex and multiplex rRT-PCR using six SARS-CoV-2 RNA-103 positive samples (Figure 1 ) and observed good correlation between the targets. However, the 104 C q values of N_Sarbeco showed an average delay of 6.4 cycles (a minimum of 5.5 to a 105 maximum of 7.5). Similar results for C q values were obtained using simplex NIID-N, multiplex 106 NIID-N and multiplex E_Sarbeco. These results indicate that the sensitivity of NIID-N was not 107 decreased by multiplexing, and E_Sarbeco and NIID-N had equal sensitivity. We also tested 108 another nucleic acid extraction kit (High Pure Viral RNA Kit, Roche). Although the C q values 109 were slightly higher than those obtained using a Maxwell RSC Viral Total Nucleic Acid 

The SARS-CoV-2 test results obtained using the novel multiplex rRT-PCR are shown in Figure   115 2. Twenty-four clinical specimens were analyzed. Four positive and twenty negative results We developed a multiplex rRT-PCR methodology for the molecular diagnosis of COVID-19.

The sensitivity of RT-PCR is affected by various factors, such as the nucleic acid extraction 5 133 method, the one-step RT-PCR reagent, and the primer/probe sets. Optimization experiments 134 are required prior to using this methodology in clinical settings. 135 Current results indicate that: 1) the NIID-N set is slightly more sensitive than E_Sarbeco; 2) 136 NIID-N and E_Sarbeco are a good combination for detecting two regions of the SARS-CoV-2 137 genome with high sensitivity; and 3) the human ABL1 gene is useful as an IC for checking the 138 qualities of the specimen, nucleic acid extraction step, and RT-PCR amplification. Therefore, 139 we demonstrated that this highly sensitive multiplex rRT-PCR methodology can detect SARS-140 CoV-2.

The human ABL1 gene is recommended as an IC gene for the molecular diagnosis and 142 monitoring for minimal residual disease detection [7] . Many molecular diagnostic laboratories 143 use this primer/probe set. We did not use the ACTB and GAPDH genes, which are also 144 frequently used as ICs, because they are more abundantly expressed than the ABL1 gene [9].

In multiplex PCR, the amplification of highly abundant targets may prevent the amplification 146 of less abundant targets, possibly lowering the sensitivity of target detection.

The background of E_Sarbeco was slightly increased in later cycles. We judged these signals 148 as negative results because: 1) reproducibility was poor (negative upon retesting with the 149 same assay), 2) NIID-N did not show positive results, although it has equal sensitivity to 

Since these results may lead to false-positive results, care must be taken when judging weak 153 positive signals.

The SARS-CoV-2 pandemic has resulted in a shortage of reagents related to the molecular 155 diagnosis. The proposed multiplex rRT-PCR methodology will enable highly sensitive 156 detection of SARS-CoV-2 RNA, reducing reagent use and cost, and time required by clinical

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Six samples were analyzed by simplex rRT-PCR

Simplex rRT-PCR with 205 NIID-N was used as a reference (y axis). The dashed line indicates the y=x line

Results of SARS-CoV-2 detection using the novel multiplex rRT-PCR method. CTRL-C and 209

included 400 copies/reaction of SARS-CoV-2 RNA and 25 copies/reaction of 210 synthetic RNA containing the N region, respectively (red solid lines). The dotted (green) and 211 dashed (blue) lines indicate negative and positive results for the clinical specimens