key: cord-0998122-0w7x177a
authors: Muraoka, M.; Tanoi, Y.; Tada, T.; Mizukoshi, M.; Kawaguchi, O.
title: Quickly And Simply Detection For Coronavirus Including SARS-CoV-2 On The Mobile Real-Time PCR Device And Without RNA Extraction
date: 2020-08-07
journal: nan
DOI: 10.1101/2020.08.06.20168294
sha: 2b2976d4cd83243aaeb221c1888bd28aab84a0c2
doc_id: 998122
cord_uid: 0w7x177a

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported to WHO as an outbreak in Wuhan City, Hubei Province, China on end of 2019, afterwards epidemic in many countries, and pandemic on the worldwide in 2020. Usually detection of coronavirus including SARS-CoV-2 was detected by real-time RT-PCR method, but it must be long time that RNA is treated by extraction, concentration and purification, and detected by RT-PCR method. We modified various methods, of which evaluated if each method is short and simple enough. In one point of the evaluations, real-time RT-PCR could be finished in very short time with using mobile real-time PCR device PCR1100 (Nippon Sheet Glass Co. Ltd.). It was able to detect positive control RNA for 20 minutes by each method according to the National Institute of Infections Disease in Japan (NIID), and less than 13.5 minutes according to the Centers for Disease Control and Prevention in USA (CDC). In another point of the evaluations, surprisingly, Human coronavirus 229E, which was substituted for SARS-CoV-2, could be detected in crude state without treatment in advance of RNA. As that was, it was possible to detect coronavirus with direct RT-PCR. Therefore, it might eliminate wasteful time, avoid secondary infection and risk of contamination. In light of the above two points, SARS-CoV-2 might be detected more quickly and more simply. With using this mobile real-time PCR, these methods should be suitable for not only SARS-CoV-2 but also other various viruses and might save time compared to earlier detection methods.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a cause of coronavirus disease 2019 (COVID-19) outbreak, of which WHO declared pandemic on March 11, 2020 [1] . was firstly reported to WHO as an outbreak in Wuhan City, Hubei Province, China on December 31, 2019, afterwards has rapidly spreading [2] . SARS-CoV-2 included, human infectious coronaviruses have been known seven types. Four of the seven types (HCoV-229E, HCoV-HKU1, HCoV-OC43, and HCoV-NL63) are included among the known causes of common cold. Whereas, other three human coronaviruses (the Middle East respiratory syndrome coronavirus [MERS-CoV], SARS-CoV, SARS-CoV-2) are highly pathogenic and can cause severe diseases presented as acute respiratory distress syndrome (ARDS) characterized by dysfunctional immune responses and severe pulmonary injury [3] [4] . The SARS-CoV-2 virus is genetically closely related to SARS-CoV, but which is less deadly and far more transmissible than MERS-CoV or SARS-CoV [5] . Therefore, it has been needed with rapidity and on site to detect and monitor. Routine confirmation of SARS-CoV-2 is based on detection of unique sequences of virus RNA by nucleic acid amplification tests (NAAT) such as real-time reverse-transcription polymerase chain reaction (RT-PCR). The viral genes targeted so far include the nucleocapsid (N), envelop (E), RNAdependent RNA polymerase (RdRP), spike (S), and open reading frame 1 a/b (Orf1a/b), each of which primer and probe is various sequences [6] [7] [8] [9] [10] [11] . We recognize that the performance characteristics of PCR methods can vary with reagents, PCR programs, and devices. Therefore, if devices or reagents changed, with simpler and faster, it might be possible to detect.

And, in quantitative RT-PCR, RNA extraction is required, followed by transcription, amplification and detection. Current RT-PCR typically require more than 60 minutes for RNA extraction, concentration and purification, and a further 90 minutes for transcription and amplification [12] . It is difficult to handle RNA not to be damaged and lost, possible to infect secondary and needed long time to detect. This might be due to the delay of the initial introduction of RT-PCR detection for SARS-CoV-2 in various places.

Already reported rapid detection of SARS-CoV-2 with using mobile real-time PCR device PCR1100 [10], we suggest "simpler and faster" method of RT-PCR in this paper as follows.

Based on previously reported protocols of NIID [6] [7] and CDC [8] [9] , and detection of HCoV-229E [13] , PCR primers and probes for real-time RT-PCR detection were selected to target each N gene, and synthesised (Fasmac Co. Ltd.) ( The above, exposure to HCoV-229E is low-risk for healthy adults. The HCoV-229E virus is easily accessible for the biosafety level 2 (BSL2) laboratory. Therefore, HCoV-229E may be a good initial model for the evaluation against other coronaviruses, such as SARS-COV-2, the coronavirus that causes COVID-19 [14] . There we substituted HCoV-229E for SARS-2-CoV in direct RT-PCR. Human coronavirus 229E (HCoV-229E, VR-740™) was bought by certified non-profit organization biomedical science association (BMSA, Japan) from American Type Culture Collection (ATCC, USA), afterwards, in cooperation of BMSA, it was able to be used. Because to proliferate HCoV-229E, it was infected with MRC-5 cells (CCL-171™, ATCC), and cultured with EMEM medium (30-2003™, ATCC) included 2% FBS (ATCC® 30-2020™) for 5~8 days in 5% CO2 at 35 °C. When virus titer was grown up 8.9 x 10^6 Median tissue culture infectious dose (TCID50/mL), only that supernatant without MRC-5 cells was stored at -80 °C before the assay. The supernatant included HCoV-229E was thawed in the assay, not treated in advance and used with crude as it was. To assess detection value of PCR1100 relative with titer of virus, the crude sample was applied 10fold serial dilution with 10mM Tris-HCl (pH8.0, the above).

RT and PCR were carrying out in the one-step with using mobile real-time PCR device PCR1100 for all tests of this study. Briefly describe the composition of RT-PCR reagent, 1 μL of a sample (the crude or positive control RNA) was amplified in a 17 μL reaction solution containing 1x Reaction Buffer, 0.25 μL of RT Enzyme Mix, 1.0 μL of DNA Polymerase (THUNDERBIRD® Probe Onestep qRT-PCR Kit, TOYOBO Co. Ltd., Japan), each final concentration of primer and probe for target detection (Table 2 .). With preliminary tested, each composition optimized (data not shown). The RT-PCR conditions applied in this study were programmed as follows: RT incubation and enzyme activation were serially performed at 50 °C for 150 seconds, at 95 °C for 15 seconds respectively. Afterwards, for SARS-CoV-2 against NIID version: thermal cycling was then performed at 95 °C for 3.5 seconds (denaturation), and at 60 °C for 16 seconds (annealing and amplification) for fifty cycles. For SARS-CoV-2 against CDC version: thermal cycling was then performed at 95 °C for 3.5 seconds (denaturation), and at 60 °C for 7 seconds (annealing and amplification) for fifty cycles. For HCoV-229E: thermal cycling was then performed at 95 °C for 3.5 seconds (denaturation), and at 60 °C for 8 seconds (annealing and amplification) for fifty cycles. With preliminary tested, each condition was optimized (data not shown).

Incidentally, the procedure of PCR1100 was according to previously reported [15] .

In NIID version, both No.1 and No.2 could be simultaneously detected with multichannel ( Fig.1.) .

As the result previously reported [7] , No.2 was detected more sensitive than No.1, possible to determine 10 copies too. Each Ct value (detected on the PCR1100) was depended on the rate of dilution (No.1:R 2 =0.9982, No.2:R 2 =0.9969). When simultaneously detecting with two kinds of primer / probe sets, accuracy of results could be provided with higher reliability. It took only about 20 minutes per detection on the PCR1100 with using one step RT-PCR reagent (THUNDERBIRD® Probe One-step qRT-PCR Kit). So, it could be to detect 10 copies of positive control RNA only in 20 minutes.

In CDC version, also, CDC No.1 and CDC No.2 could be detected with multichannel (Fig.2. ). Both No.1 and No.2 were detected in same sensibility, and possible to detect 10 copies too. Furthermore, each Ct value (detected on the PCR1100) was depended on the rate of dilution (No.1:R 2 =0.9996, No.2:R 2 =0.9983). As the same of NIID version, when simultaneously detecting with two kinds of primer / probe sets, accuracy of results could be provided with higher reliability. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 7, 2020. .

And it took only about 13.5 minutes per detection on the PCR1100 with using the same one step RT-PCR reagent, that was even faster compared with NIID version. It could detect with at least10 copies of positive control RNA, besides it took only in 13.5 minutes.

Crude sample of HCoV-229E was applied 10-fold serial dilution, afterwards without treatment in advance and with one step RT-PCR reagent (THUNDERBIRD® Probe One-step qRT-PCR Kit) was tested in the RT-PCR on the mobile real-time PCR device PCR1100. As the result, without treatment for extraction, concentration and purification of RNA, it was possible to detect HCoV-229E at least 8.9 x 10 2 TCID50/mL (Fig.3.) . Furthermore, each Ct value (detected on the PCR1100) was depended on the rate of dilution, that is, virus titer (R 2 =0.9949). Ct value might be relatively indicated to virus titer.

Also, it took only about 13 minutes per detection on the PCR1100 with using one step RT-PCR reagent the same above of SARS-CoV-2. On another real-time PCR device using normal two steps RT-PCR, it needed more than 90 minutes. Therefore, it would be able to omit more than 135 minutes (60 minutes for RNA treatment, 75 minutes for RT-PCR) in total in detection of RNA virus compared with other current methods.

Previously reported [10] [15] [16] , with using the mobile real-time PCR device PCR1100, it could be to detect unique strain rapidly, and sensibility and specificity are equivalent to other devices. But it would take 15 minutes at least to conclude RT-PCR for detection of unique RNA in any case. Furthermore, it was necessary for RNA in advance to adjust with extraction, concentration and purification.

First, this study addressed it to optimize each composition and PCR program according to previously reported [7] [10], a part of which modified to quickly carry out RT-PCR for detection of SARS-CoV-2 with using the mobile real-time PCR device PCR1100. Especially with the unit of reverse transcriptase enzyme decreased and the time of reverse transcription shorted, sensibility of detection was improved (data not shown). And, as one step RT-PCR reagent, THUNDERBIRD Probe One-step qRT-PCR Kit (TOYOBO) was used, of which was possible and efficiently to carry out RT and probe' real-time PCR simultaneously. This time, NIID and CDC version were selected for detection of SARS-CoV-2, both of which were unique for N-gene of SARS-CoV-2 and used in general [7] [8] [9] . As a result, in the NIID version No.2, CDC version No.1 and No.2, it was possible quantitatively to detect 10 copies or more of positive control RNA with using mobile real-time PCR device PCR1100. However, in the NIID, No.1 was able to detect only 100 copies or more, so No.2 was more sensitive than No.1 by 10 times. These results were about equal to previously reported [7] [10], that is, it might mean this method to be right adequate to detect positive control RNA for SARS-CoV-2. Furthermore, in both of CDC version by multichannel method, RT-PCR run could be finished in less than 14 minutes. That is, after RNA finished of extraction from each sample, this device would be quickly able to discriminate presence of target. This faster RT-PCR might be caused by combination of PCR1100 with RT-PCR reagent, of which was possible and efficiently to carry out RT and PCR simultaneously. But regretfully, in our situation unable to deal with SARS-CoV-2, this method couldn't be demonstrated possible or not for genome RNA as reported previously [7] [10] . And primer / probe sets unique for gene of SARS-CoV-2 has been variously developed till now, so we would like to investigate about those sets if RT-PCR is able to be faster to more in the PCR1100. Second, we tried it to modify total system to detect SARS-CoV-2 easier. When treatment in advance for RNA (mainly extraction, concentration and purification) it was required more than 60 minutes [12] [13] [17] . Therefore, in this study, we tried it to carry out RT-PCR without treatment of RNA in is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20168294 doi: medRxiv preprint advance, so that, direct RT-PCR. We tested for direct RT-PCR, in that was used THUNDERBIRD® Probe One-step qRT-PCR Kit as one step RT-PCR reagent. With above, this reagent was possible and efficiently to carry out RT and probe' real-time PCR simultaneously, furthermore as reason, described possible to NAAT from crude samples [18] . And because of our situation unable to deal with SARS-CoV-2, therefore we substituted HCoV-229E, low-risk for healthy, for SARS-2-CoV in direct RT-PCR this time. As a result, surprisingly, without treatment for extraction, concentration and purification of RNA, it was possible to detect HCoV-229E at least 8.9 x 10 2 TCID50/mL Furthermore, Ct value could be relatively to virus titer. This time, Lower Limit of Detection (LoD) couldn't show accurately because failed to measure less than 8.9 x 10 2 TCID50/mL. But as approximate curve shown (y=-4.004x+46.61), the LoD is supposed to less than 1 TCID50/mL. Also, it took only about 13 minutes per detection on the PCR1100 with using one step RT-PCR reagent the same above of SARS-CoV-2. Therefore, it would be able to omit more than 135 minutes (60 minutes for RNA treatment, 75 minutes for RT-PCR) in total in detection of SARS-CoV-2 compared with other current methods. Already some reported direct RT-PCR for SARS-CoV-2, it was necessary for especial reagent and additional treatment duration to direct RT-PCR [12] [19] [20] [21] [22] . So, we could suggest novel method in this paper, in which used PCR1100 device and commercial one step RT-PCR reagent only. However, our method must be assessed in real SARS-CoV-2, further validated using clinical specimens, which contained nasopharyngeal swabs and sputum. Furthermore, real-time RT-PCR reliable in saliva reported [23] [24], its specimen must be assessed too. On the other hand, if low virus in air could be collected, it might be able to detect quickly and easily on site by our novel method, and the result be following prevent from droplet infection.

Although the other four community-acquired HCoVs (229E, OC43, HKU1, NL63) are a common cause of common cold only, they are thought to cause pandemics and major outbreaks of probably more severe respiratory diseases when they initially crossed species' barriers to infect humans decades and centuries ago [3] [25] . That is, it would be possible new coronaviruses to transfer again, therefore we are exposed to dangerous forever. On that time, the mobile real-time PCR could be it save time compared to earlier detection methods. And we think that methods suggested in this paper should be suitable for various strains and it to be useful.

When each sample was assessed in the real-time RT-PCR, a ten-fold dilution series of each was indicated with high relativity to Ct value on the mobile real-time PCR device PCR1100 (R 2 =0.99). Table 1 . Selected primers and probes for the real-time RT-PCR of SARS-CoV-2 NIID and CDC, and HCoV-229E *R= A or G, **Y= C or T is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 7, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20168294 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20168294 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20168294 doi: medRxiv preprint

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Development of Genetic Diagnostic Methods for nCoV-2019 in Japan

Centers for Disease Control and Prevention "2019-Novel Coronavirus (2019-nCoV) Real-time rRT-PCR Panel Primers and Probes

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TOYOBO Life Science Raw Material Catalog

DIRECT RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWABS WITHOUT AN RNA EXTRACTION STEP" bioRxiv

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We thank Dr. Kazuya Shirato and Dr. Tsutomu Kageyama at the National Institute of Infections Disease in Japan (NIID) for positive control or technical comments, and Takashi Fukuzawa in an exemployee at Nippon Sheet Glass Co. Ltd. for technical comments or experimental help.

The authors declare that no competing interests exist. While M. Muraoka, T. Tanoi, T. Tada, and O. Kawaguchi were employed at Nippon Sheet Glass at the time of this study, in where all did not create a competing interest. Further, Nippon Sheet Glass had no involvement in this study.