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

Abstract

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.

Natural spring water gargle samples as an alternative to nasopharyngeal swabs for SARS‐CoV‐2 detection using a laboratory‐developed test

“…Indeed, some studies suggested that pretreatment of samples with PK increased the sensitivity of direct rRT‐PCR for SARS‐CoV‐2 detection, 28,29 particularly in low positive samples with high C t values 13 . It has been suggested that PK could degrade or inactivate RNAses, allowing to purify a better quality of RNA 30 . Another study showed that sputum specimens pretreated with PK for homogenization before nucleic acid extraction for RT‐PCR had a higher detection rate than those pretreated with saline only.…”

Development and Testing of a Low-Cost Inactivation Buffer That Allows Direct Sars-Cov-2 Detection in Saliva

“…Saliva is a source of RNases that may degrade the SARS-CoV-2 RNA genome, compromising its detection by RT-qPCR [18]. To overcome this situation, we added the thermostable RNase inhibitor Poly-Vinyl Sulfonic Acid (PVSA) to the PKT buffer (hereafter PKTP), a low cost-reagent that can enhance RNA preservation at low concentrations [19].…”

“…This buffer is compatible with a heat-inactivation step that has been previously demonstrated to eliminate SARS-CoV-2 in saliva [13, 22], minimizing the biohazard risks of handling the samples. However, RNA degradation is a concern when heating or storing samples, as saliva contains thermo-stable RNases that may compromise the diagnosis [18]. Our own data showed that storage of saliva resulted in low molecular weight RNA molecules indicative of degradation (Fig.…”

Point of care testing for detection of coronaviruses including SARS-CoV-2 from saliva without treating RNA in advance

“…As a result, serial dilution rate with the saliva sample was highly correlated to Ct value with equivalent to Tris buffer. As several studies have reported, salivary RNA is prone to degradation by RNases and other nucleases present in saliva [27, 28]. Hence, it was concluded that this method made it possible to detect virus in saliva without treating in advance (extraction, purification, concentration, etc.…”