Validation of saline, PBS and a locally produced VTM at varying storage conditions to detect the SARS-CoV-2 virus by qRT-PCR

Abstract

Coronavirus Disease-2019 tests require a Nasopharyngeal (NP) and/or Oropharyngeal (OP) specimen from the upper airway, from which virus RNA is extracted and detected through quantitative reverse transcription-Polymerase Chain Reaction (qRT-PCR). The viability of the virus is maintained after collection by storing the NP/OP swabs in Viral Transport Media (VTM). We evaluated the performance of four transport media: locally manufactured ("REVITAL") Viral Transport Media (RVTM), Standard Universal Transport Media (SUTM), PBS and 0.9% (w/v) NaCl (normal saline). We used laboratory cultured virus to evaluate: i) viral recovery and maintaining integrity at different time periods and temperatures; ii) stability in yielding detectable RNA consistently for all time points and conditions; and iii) their overall accuracy. Four vials of SARS-CoV-2 cultured virus (2 high and 2 low concentration samples) and 1 negative control sample were prepared for each media type (SUTM, RVTM, PBS and normal saline) and stored at the following temperatures, -80°C, 4°C, 25°C and 37°C for 7 days. Viral RNA extractions and qRT-PCR were performed at 1, 2, 3, 4 and 7 days after inoculation with the cultured virus to assess virus stability and viral recovery. Ct values fell over time at 25°C and 37°C, but normal saline, PBS, RVTM and SUTM all showed comparable performance in maintaining virus integrity and stability allowing for the detection of SARS-CoV-2 RNA. Overall, this study demonstrated that normal saline, PBS and the locally manufactured VTM can be used for COVID-19 sample collection and testing, thus expanding the range of SARS-CoV-2 viral collection media.

Fig 1. An illustration showing virus dilutions in different media types and different virus inoculation temperatures.

Each media type was made up of 2 tubes inoculated with high virus concentrations, 2 tubes of low virus concentrations and 1 negative control (no viral inoculum) tube, generating a total of 5 tubes stored at the following temperatures: -80°C, 4°C, 25°C (room temperature) and 37°C and subsequently used to extract RNA for qRT-PCR on days 1, 2, 3, 4 and 7. Thus, each media type consisted of a total of 80 tubes, that were subsequently extracted for RNA to conduct the qRT-PCR.

Fig 2. Mean Ct values for the four media types over time (in days) at -80ºC, 4ºC, room temperature (25ºC) and 37ºC stratified by concentration.

A Ct>36 or 37 indicates a negative result. The gaps in temperatures -80°C and 4°C indicate the negative samples, these were samples with Ct<36, which were excluded from the analyses. Each data point is the mean of 4 samples, the 2 high and 2 low concentration samples assayed in duplicate and the negative control sample data points are the mean of the duplicates.

Fig 3. Correlations between Ct values from the 4 media types.

(A) Scatter plots for Ct values at low concentration. (B) Scatter plots for Ct values at high concentration. The line of best fit was estimated using a Deming regression model.