Stability of SARS-CoV-2 in Phosphate-Buffered Saline for Molecular Detection.

LETTER

RNA viruses often require “cold chains” of transportation to prevent the breakdown of genetic material. The logistics of getting samples from the patient to a diagnostic laboratory, sometimes thousands of miles away from the original collection site, can be complex and resource intensive (1, 2). Nucleic acid degradation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can compromise the accuracy of molecular detection methods. It has been demonstrated that nasopharyngeal specimens containing SARS-CoV-2 can be stored in phosphate-buffered saline (PBS) as a substitute for viral transport medium (VTM) for up to 7 days (3). Here, we evaluate the stability of differing viral loads of SARS-CoV-2 over 28 days stored at room temperature, 4°C, –20°C, or –80°C.

We used the first SARS-CoV-2-positive nasopharyngeal swab detected in our laboratory for spike-in material. PBS spiked with this SARS-CoV-2 specimen was stored in quadruplicates and divided into two concentrations, namely, 5,000 to 10,000 copies/ml (high titer) and 500 to 1,000 copies/ml (low titer), as determined by droplet digital reverse transcriptase PCR (RT-PCR). Nucleic acids were isolated on the Roche MagNA Pure96 system (Basel, Switzerland). Qualitative RT-PCR (qRT-PCR) was performed on 448 samples using our CDC-based laboratory-developed test, as described previously (4, 5).

For the high concentration of SARS-CoV-2, regardless of storage conditions, 100% of samples were detected by qRT-PCR through day 28. At room temperature, median cycle threshold (C) values for lower titers for both N1 and N2 targets remained consistent through day 28, fluctuating less than 1 median C (Table 1). For lower concentrations of virus, storage at room temperature was associated with reductions of positivity beginning at day 7, and by day 28, 0% of samples were detected for N1. Storage at room temperature was the least stable of all environmental conditions tested, with 54.2% of negative PCR results.

TABLE 1

Median C values for N1 and N2 targets

Storage condition by target and titer	Median CT value
	Day 0	Day 1	Day 2	Day 7	Day 14	Day 21	Day 28
N1
5,000–10,000 copies/ml
Room temp	31.4	31.0	31.5	31.2	31.6	31.4	31.3
4°C	30.8	30.9	31.3	31.5	31.7	31.5	31.4
−20°C	30.9	31.4	31.7	31.2	31.7	31.8	31.8
−80°C	30.7	31.1	31.4	31.0	31.0	30.9	31.8
500–1,000 copies/ml
Room temp	33.4	36.8	36.3	36.1 (3/4)b	35.8 (3/4)	35.2 (1/4)	NDET (0/4)
4°C	34.4	34.9	35.1	35.9	35.9	35.8 (3/4)	36.5
−20°C	34.2	35.2	36.0	36.5	37.5 (1/4)	37.4	37.3 (3/4)
−80°C	35.1	34.4	35.3	34.9	36.2	36.4	35.3
N2
5,000–10,000 copies/ml
Room temp	30.6	30.4	30.9	30.9	31.1	31.1	31.2
4°C	30.4	30.2	30.5	30.6	31.3	30.6	31.1
−20°C	30.6	30.5	30.9	30.6	31.6	30.9	31.2
−80°C	30.6	30.5	30.6	30.6	30.6	30.5	31.2
500–1,000 copies/ml
Room temp	32.8	35.0	35.8	36.6 (3/4)	37.0	38.3 (2/4)	38.3 (3/4)
4°C	33.8	34.2	34.7	34.9 (3/4)	36.0	35.4	36.4
−20°C	33.7	35.3	36.0	37.8 (3/4)	36.7	36.3 (3/4)	37.0 (3/4)
−80°C	34.4	33.7	34.8	35.7	35.8	34.7	35.9

C, cycle threshold; NDET, not detected. N1 and N2 are amplicons within the nucleocapsid gene of SARS-CoV-2.

Parentheticals denote the number of samples that were detected at a condition only if the 4 replicates were not all detected.

At 4°C, there was minimal change in Cs over time at the higher viral concentration. For lower titers, Cs increased by 2.1 Cs for N1 and 2.6 Cs for N2 over the 28 days. At –20°C, lower titers of virus fluctuated slightly more, increasing by ≥3 Cs. Storage of SARS-CoV-2 in PBS at –20°C was the second least stable condition, accounting for 37.5% of negative PCR results. Storage at –80°C showed the greatest stability, with all samples detected throughout the 28 days and ≤1.5 median Cs for both N1 and N2 targets.

Here, we show that the stability of SARS-CoV-2 can be maintained at 4°C for up to a month when –80°C storage is not available (6, 7). At viral loads of >5,000 copies/ml—corresponding to >75% of positive samples recovered in our clinical lab to date—different storage temperatures did not have a substantial impact on our ability to detect SARS-CoV-2 when stored in PBS.