key: cord-340410-s9haq8y1
authors: Fukumoto, Tatsuya; Iwasaki, Sumio; Fujisawa, Shinichi; Hayasaka, Kasumi; Sato, Kaori; Oguri, Satoshi; Taki, Keisuke; Nakakubo, Sho; Kamada, Keisuke; Yamashita, Yu; Konno, Satoshi; Nishida, Mutsumi; Sugita, Junichi; Teshima, Takanori
title: Efficacy of a novel SARS-CoV-2 detection kit without RNA extraction and purification
date: 2020-06-26
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2020.06.074
sha: 
doc_id: 340410
cord_uid: s9haq8y1

Abstract Rapid detection of SARS-CoV-2 is critical for the diagnosis of coronavirus disease 2019 (COVID-19) and preventing the spread of the virus. A novel “2019 Novel Coronavirus Detection Kit (nCoV-DK)” halves detection time by eliminating the steps of RNA extraction and purification. We evaluated concordance between the nCoV-DK and direct PCR. The virus was detected in 53/71 (74.6%) and 55/71 (77.5%) by the direct PCR and nCoV-DK, respectively, with overall concordance rate of 94.4%: 95.2% in nasopharyngeal swab, 95.5% in saliva, and 85.7% in sputum. The nCoV-DK effectively detects SARS-CoV-2 in all types of the samples including saliva, while reducing time required for detection, labor, and risk of human error.

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Takanori Teshima, M.D., Ph.D.

Department of Hematology, Hokkaido University Faculty of Medicine N15 W7, Sapporo, E-mail: teshima@med.hokudai.ac.jp

Rapid and accurate detection of SARS-CoV-2 is critical for the prevention of outbreaks of coronavirus disease 2019 in communities and hospitals. The diagnosis of COVID-19 is made by real-time quantitative PCR (RT-qPCR) testing of specimens collected by nasopharyngeal swabs (Wang et al., 2020 , Zou et al., 2020 . However, swab sample collection poses a risk of viral transmission to healthcare workers. Self-collecting saliva reduces a risk of health care workers. We and others have shown efficacy of saliva J o u r n a l P r e -p r o o f 4 as a diagnostic tool (Azzi et al., 2020 , Iwasaki et al., 2020 , Williams et al., 2020 , Wyllie et al., 2020 .

The 2019 Novel Coronavirus Detection Kit (nCoV-DK, Shimadzu Corporation, Kyoto, Japan) eliminates the steps of RNA extraction and purification by using the Ampdirect TM technology (Nishimura et al., 2010) , thus significantly reducing the time required for sample preparation and PCR detection from more than 2 hours to about 1 hour. In addition, the risk of human error during RNA extraction can be reduced. However, it remained to be elucidated whether saliva samples could be applied to the nCoV-DK, since saliva has high RNase (Pandit et al., 2013) . We herein compared efficacy of the nCoV-DK with the direct PCR method requiring RNA extraction and purification.

Samples were collected from 9 patients with COVID-19, as previously described (Iwasaki et al., 2020) . A total of 71 frozen stock samples were available from these patients, with a median of 8 samples (range, 2-15) per patient. This study was approved by the Institutional Ethics Board and informed consent was obtained from all patients. 

Agreement between the two methods was assessed using Cohen's Kappa. Pearson's correlation coefficient test was performed to identify the relation of the Ct values between the methods. Statistical analyses were performed with EZR (Jichi Medical University, Saitama, Japan). P-value of 0.05 was the cutoff for statistical significance.

We first examined whether the freeze-thaw step could affect the availability of viral RNA for detection. The nCoV-DK PCR was performed in 3 fresh samples and the corresponding freeze-thaw specimens. Ct values did not change significantly after the freeze-thaw steps (Supplementary Figure 1) .

We then evaluated the viral detection rates in 71 specimens. The virus was detected in 53 (74.6%) fresh samples by the direct PCR and in 55 (77.5%) of the corresponding frozen J o u r n a l P r e -p r o o f samples by the nCoV-DK (Table 1 ). The overall concordance rate of the virus detection between the two methods was 94.4% (95% CI, ). Interrater reliability of the two methods was strong (=0.85), as judged by Cohen's kappa analysis. Concordance rates were 95.2% (95% CI, 83.8-99.4), 95.5% (95% CI, 77.2-99.9), 85.7% (95% CI, 42.1-99.6) in nasopharyngeal swab, saliva, and sputum samples, respectively. Figure 2 shows a scatter plot presenting a comparison of Ct values in each sample between the two methods. There was a strong correlation between the two methods (r = 0.837, 95%CI = 0.736-0.902, P < 0.01). Significant correlations were also demonstrated in each sample type (Swab, r = 0.82, 95%CI = 0.673-0.905, P < 0.01; Saliva, r = 0.818, 95%CI = 0.507-0.94, P < 0.01; Sputum, r = 0.945, 95%CI = 0.574-0.994, P < 0.01).

We demonstrate that a novel SARS-CoV-2 detection kit nCoV-KD is as effective as the direct PCR in detecting SARS-CoV-2 in all types of the samples. Particularly, it should be noted saliva is a reliable tool to detect the virus by the nCoV-KD even without process of RNA extraction and purification. There are some discordant results between the two methods. The virus was detected only by the direct PCR in one sample, while the virus was detected only by the nCoV-DK in 3 samples. It is unclear whether these are false-positive or J o u r n a l P r e -p r o o f 

Saliva is a detect SARS-CoV-2

Comparison of SARS-CoV-2 detection in nasopharyngeal swab and saliva

Detection of noroviruses in fecal specimens by direct RT-PCR without RNA purification

High-yield RNA-extraction method for saliva

Consistent detection of 2019 novel coronavirus in saliva

Detection of SARS-CoV-2 in Different Types of Clinical Specimens

Saliva as a non-invasive specimen for detection of SARS-CoV-2

Saliva is more sensitive for SARS-CoV-2 detectionin COVID-19 patients than nasopharyngeal swabs

This work was in part supported by grants from Japan Medical Association Policy Research Organization (JMARI).

The authors declare that they have no competing interests.