RNase in the saliva can affect the detection of severe acute respiratory syndrome coronavirus 2 by real-time one-step polymerase chain reaction using saliva samples

Nishibata, Yuka; Koshimoto, Shota; Ogaki, Kenta; Ishikawa, Erika; Wada, Kosuke; Yoshinari, Miku; Tamura, Yuto; Uozumi, Ryo; Masuda, Sakiko; Tomaru, Utano; Ishizu, Akihiro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a single-stranded RNA virus that causes coronavirus disease 2019, which spread worldwide immediately after the first patient infected with this virus was discovered in Wuhan, China, in December 2019. Currently, polymerase chain reaction (PCR) specimens for the detection of SARS-CoV-2 include saliva, nasopharyngeal swabs, and lower respiratory tract-derived materials such as sputum. Initially, nasopharyngeal swab specimens were applied mainly to the PCR detection of SARS-CoV-2. There was a risk of infection to healthcare workers due to coughing or sneezing by the subjects at the time of sample collection. In contrast, saliva specimens have a low risk of droplet infection and are easy to collect, and their application to PCR testing has been promoted. In this study, we have determined the detection limit of SARS-CoV-2 in saliva samples and examined the effects of storage temperature and storage time of saliva samples on the PCR detection results. As a result, 5 × 10(3) copies of SARS-CoV-2 could be detected in 1 mL phosphate-buffered saline, whereas 5 × 10(4) copies of SARS-CoV-2 were needed in 1 mL saliva to detect the virus by real-time one-step PCR. Interestingly, SARS-CoV-2 (5 × 10(3) copies/mL) could be detected in saliva supplemented with an RNase inhibitor. Concerning the saliva samples supplemented with an RNase inhibitor, the optimal temperature for sample storage was -20 °C, and PCR detection was maintained within 48 h without problems under these conditions. These finding suggest that RNase in the saliva can affect the detection of SARS-CoV-2 by PCR using saliva samples.