Development of a simple and highly sensitive virion concentration method to detect SARS-CoV-2 in saliva

Abstract

Background: Controlling novel coronavirus pandemic infection (COVID-19) is a global challenge, and highly sensitive testing is essential for effective control. The saliva is a promising sample for high-sensitivity testing because it is easier to collect than nasopharyngeal swab samples and allows large-volume testing.

Results: We developed a simple SARS-CoV-2 concentration method from saliva samples that can be completed in less than 60 min. We performed a spike test using 12 ml of saliva samples obtained from healthy volunteer people, and the developed method performance was evaluated by comparison using a combination of automatic nucleic acid extraction followed by RT-qPCR detection. In saliva spike tests using a 10-fold dilution series of SARS-CoV-2, the developed method was consistently 100-fold more sensitive than the conventional method.

Conclusions: The developed method can improve the analytical sensitivity of the SARS-CoV-2 test using saliva and speed up and save labor in screening tests by pooling many samples. Furthermore, the developed method has the potential to contribute to the highly sensitive detection of various human and animal viral pathogens from the saliva and various clinical samples.

Keywords: COVID-19; Concentration; SARS-CoV-2; Semi alkaline proteinase; Virion; Virus.

Fig. 1

Developed protocol for virion concentration from saliva. SAP, Semi-alkaline proteinase. 1. Mix 12 ml of saliva with 12 ml of SAP (1:1) and keep for 15 min at room temperature. 2. After centrifugation at 4000 g for 5 min, carefully transfer 18 ml of the supernatant into a new 50-ml tube. 3. Add 15 ml of SAP to 18 ml of the supernatant, mix using a vortex and then keep at room temperature for 15 min. 4. After adding 13.2 ml of PEG-NaCl solution, mix by vortexing. 5. After centrifugation at 8000 g for 20 min, carefully discard the supernatant. 6. Add 100 μl of PBS and dissolve the invisible precipitates by pipetting and scraping with a 1-ml long tip (10 times each). 7. Place a 1-ml short tip into a 50-ml tube and vortex to completely dissolve the precipitate (supernatant residue after flushing + PBS ≒ 200 μl), and then transfer to an RNA extraction tube.

Fig. 2

Virus detection from saliva with developed and conventional methods. SAP, Semi-alkaline proteinase. Conventional method: Nucleic acid extraction of 200 μl of centrifuged supernatant comprising of saliva 50 μl + PBS or SAP 150 μl (4x dilution). Developed method: Nucleic acid extraction after a simple concentration of 12 ml of saliva. The illustrations are cited from the following sources, all used in compliance with the terms and conditions. Pipettes and Micropipettes: Irasutoya (irasutoya.com). Vortex mixer: Kagaku Irasuto (science-illust.com). 50 ml tubes: Kenkyu Net (wdb.com/kenq/illust). Automatic nucleic acid extractor: Precision System Science Co. Ltd. (pss.co.jp/).