Improved sensitivity, safety, and rapidity of COVID-19 tests by replacing viral storage solution with lysis buffer

Abstract

Conducting numerous, rapid, and reliable PCR tests for SARS-CoV-2 is essential for our ability to monitor and control the current COVID-19 pandemic. Here, we tested the sensitivity and efficiency of SARS-CoV-2 detection in clinical samples collected directly into a mix of lysis buffer and RNA preservative, thus inactivating the virus immediately after sampling. We tested 79 COVID-19 patients and 20 healthy controls. We collected two samples (nasopharyngeal swabs) from each participant: one swab was inserted into a test tube with Viral Transport Medium (VTM), following the standard guideline used as the recommended method for sample collection; the other swab was inserted into a lysis buffer supplemented with nucleic acid stabilization mix (coined NSLB). We found that RT-qPCR tests of patients were significantly more sensitive with NSLB sampling, reaching detection threshold 2.1±0.6 (Mean±SE) PCR cycles earlier then VTM samples from the same patient. We show that this improvement is most likely since NSLB samples are not diluted in lysis buffer before RNA extraction. Re-extracting RNA from NSLB samples after 72 hours at room temperature did not affect the sensitivity of detection, demonstrating that NSLB allows for long periods of sample preservation without special cooling equipment. We also show that swirling the swab in NSLB and discarding it did not reduce sensitivity compared to retaining the swab in the tube, thus allowing improved automation of COVID-19 tests. Overall, we show that using NSLB instead of VTM can improve the sensitivity, safety, and rapidity of COVID-19 tests at a time most needed.

COVID-19 PCR test performance on samples stored at ambient temperature

“…Previous studies on SARS-CoV-2 revealed that the Ct values were increased by time at different storage temperatures ( Erster et al, 2021 ; Kim et al, 2021 ; Engelmann et al, 2021 ). Although it was not statistically significant, in our study this was the case for human RNase P gene Ct values; on contrary the Ct values for viral genes ORF and N showed a decreasing trend.…”

Effective bubble-based testing for SARS-CoV-2 using swab-pooling

“…In all 24 cases where more than one positive individual was part of the pool, the pool tested positive. preservative), as previous works showed an advantage in sensitivity to lysis buffer over VTM [15]. This higher sensitivity of the pool may result from the accumulation effect of viral RNA from several positive samples in the pool, each having a viral load that was below detection level when tested individually, but rendering the pool positive.…”

“…Simple and fast detection methods are available, but the gold standard is still qRT-PCR of nasal or nasopharyngeal swabs, which typically takes several hours and is carried out in an authorized laboratory [7,8]. Several approaches were developed to increase the throughput of qRT-PCR testing, e.g., sample pooling, which enables a shorter turnaround time with a slight reduction in test sensitivity [9][10][11][12][13][14], as well as using lysis buffer for swab collection instead of viral preservation medium [15]. While numerous studies were published on sample pooling [e.g., [9][10][11][12][13]], only a few studies were published on swab-pooling [16][17][18][19][20], with only one large-scale study [21].…”

The Effect of Pooling on the Detection of the Nucleocapsid Protein of SARS-CoV-2 with Rapid Antigen Tests

“…We expect the uncertainty associated with sampling by oro- and nasopharyngeal swab probing to be reduced when including saliva, as in our preferred procedure. The uncertainty may be reduced further with improvements over standard swabs [ 28 ] or extraction buffer composition, as discussed for tests detecting nucleic acids [ 29 ]. We note that our procedure deviates from that recommended by Abbott and that a clinical study is planned to obtain more robust data from outbreaks of COVID-19.…”