Inactivation analysis of SARS-CoV-2 by specimen transport media, nucleic acid extraction reagents, detergents and fixatives

Welch, Stephen R.; Davies, Katherine; Buczkowski, Hubert; Hettiarachchi, Nipunadi; Green, Nicole; Arnold, Ulrike; Jones, Matthew; Hannah, Matthew J.; Evans, Reah; Burton, Christopher R; Burton, Jane E.; Guiver, Malcolm; Cane, Patricia A.; Woodford, Neil; Bruce, Christine; Roberts, Allen D. G.; Killip, Marian J.

doi:10.1101/2020.07.08.194613

Cited by 23 publications

(45 citation statements)

References 32 publications

(28 reference statements)

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“…Combined nose and throat swab samples are preferably resuspended in 2 ml of SAMBA SARS-CoV collection (SCoV) buffer, which is provided with the kit. SAMBA SCoV buffer contains detergent and is at a low pH; it has been shown to inactivate SARS-CoV-2 when examined using a pseudovirus luciferase assay as previously described ( 11 ) and shows a 4.8-log 10 reduction in titer when tested with tissue culture fluid containing SARS-CoV-2 ( 12 ). However, the system can be used with Remel M4RT or MWE Virocult viral transport medium (VTM) if the sample is diluted 1:2 with SCoV buffer prior to processing.…”

Section: Methodsmentioning

confidence: 99%

Performance Evaluation of the SAMBA II SARS-CoV-2 Test for Point-of-Care Detection of SARS-CoV-2

et al. 2020

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Nucleic acid amplification for the detection of SARS-CoV-2 RNA in respiratory samples is the standard method for diagnosis. The majority of this testing is centralised and therefore turnaround times of several days. Point-of-care testing with rapid turnaround times would allow more effective triage in settings where patient management and infection control decisions need to be made rapidly. Inclusivity and specificity of the SAMBA II SARS-CoV-2 Test was determined by both in silico analyses of the primers and probes and wet testing. The SAMBA II SARS-CoV-2 Test was evaluated for performance characteristics. Clinical performance was evaluated in residual combined throat/nose swabs and compared to the Public Health England real-time PCR assay targeting the RdRp gene. The SAMBA II SARS-CoV-2 Test has an analytical sensitivity of 250 cp/mL detecting two regions of the genome (Orf1ab and N). The clinical performance was evaluated in 172 residual combined nose/throat swabs provided by the Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge (CMPHL), which showed an estimated positive percent agreement of 98.9% (95% CI: 93.83-99.97) negative percent agreement of 96.4% (95%CI: 89.92-99.26) compared to testing by CMPHL. The data shows that the SAMBA II SARS-CoV-2 Test performs equivalently to the centralised testing methods with shorter turnaround time of 86-101 minutes). Point of care testing, such as SAMBA, should enable rapid patient management and effective implementation of infection control measures.

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Section: Methodsmentioning

confidence: 99%

Performance Evaluation of the SAMBA II SARS-CoV-2 Test for Point-of-Care Detection of SARS-CoV-2

et al. 2020

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“…Sixty nasopharyngeal swabs samples were collected from healthy volunteers using Rayon swab ( n = 30) or cotton swabs ( n = 30) and placed into cobas ® PCR Media tubes (Roche Molecular Systems, Inc. Basel, Switzerland) containing 4.3 mL of guanidine hydrochloride (<40%). Guanidine hydrochloride virtually inactivates all pathogens that may be present in the sample, preserves all nucleic acid of the specimen and protects the operator from contamination during sample manipulation [ 11 , 12 , 13 , 14 ]. Guanidine hydrochloride also guarantees the stability of the contrived samples, allowing us to introduce synthetic SARS-CoV-2 RNA into authentic nasopharyngeal specimens making the testing primary sample as real as possible.…”

Section: Methodsmentioning

confidence: 99%

Analytical Sensitivity and Specificity of Two RT-qPCR Protocols for SARS-CoV-2 Detection Performed in an Automated Workflow

Barra

Rita

Mesquita

et al. 2020

Genes

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WHO declared the novel coronavirus (COVID-19) outbreak a global pandemic on 11 March 2020. The establishment of standardized RT-qPCR protocols for respiratory secretions testing, as well as sharing of specimens, data, and information became critical. Here, we investigate the analytical performance of two interim RT-qPCR protocols (Charité and Centers for Disease Control (CDC)) for the qualitative detection of SARS-CoV-2 executed in a fully automated platform. Analytical specificity, PCR amplification efficiency, analytical sensitivity (limit of detection), and cross-reactivity were evaluated using contrived samples. The on-going accuracy was evaluated by retrospective analysis of our test results database (real clinical samples). N1, E, and a modified version of RdRP assays presented adequate analytical specificity, amplification efficiency, and analytical sensitivity using contrived samples. The three assays were applied to all individuals who requested the SARS-CoV-2 molecular test assay in our laboratory and it was observed that N1 gave more positive results than E, and E gave more positive results than RdRP (modified). The RdRP and E were removed from the test and its final version, based on N1 assay only, was applied to 30,699 Brazilian individuals (from 19 February 2020 to 8 May 2020). The aggregated test results available in the database were also presented.

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“…The validation of 0.9% saline and PBS as an alternative transport medium for SARS-CoV-2 testing has been done by others [ 9 , 10 ]. The downside of 0.45% saline use instead of guanidine hydrochloride is that the former does not inactivate the microorganisms that may be present in the sample, and extra cautious should be applied during the sample measurement [ 7 ]. To our knowledge, this is the first description of use of 0.45% saline as a viral transport media for SARS-CoV-2 detection.…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, we validated the sample collection in tubes containing guanidine hydrochloride. This viral transport media virtually inactivates the pathogens present in the sample and preserves the nucleic acid of the specimen [ 5 , 6 , 7 , 8 ], however, it this also ended up resulting in a short supply. It has been described that 0.9% saline and phosphate-buffered saline (PBS) are appropriated viral transport media for SARS-CoV-2 collection and testing during the shortage of commercials tubes containing a viral transport medium [ 9 , 10 ].…”

Section: Introductionmentioning

confidence: 99%

Overcoming Supply Shortage for SARS-CoV-2 Detection by RT-qPCR

Barra

Rita

Mesquita

et al. 2021

Genes

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In February 2020, our laboratory started to offer a RT-qPCR assay for the qualitative detection of severe acute respiratory syndrome coronavirus 2. A few months after the assay was released to our patients, some materials, reagents, and equipment became in short supply. Alternative protocols were necessary in order to avoid stopping testing to the population. However, the suitability of these alternatives needs to be validated before their use. Here, we investigated if saliva is a reliable alternative specimen to nasopharyngeal swabs; if 0.45% saline is a reliable alternative to guanidine hydrochloride as a collection viral transport media; the stability of SARS-COV-2 in guanidine hydrochloride and in 0.45% saline for 10 and 50 days at room temperature; and if the primers/probe concentration and thermocycling times could be reduced so as to overcome the short supply of these reagents and equipment, without a significant loss of the assay performance. We found that saliva is not an appropriated specimen for our method—nasopharyngeal swabs perform better. Saline (0.45%) and guanidine hydrochloride have a similar SARS-CoV-2 diagnostic capability as tube additives. Reliable SARS-CoV-2 RNA detection can be performed after sample storage for 10 days at room temperature (18–23 °C) in both 0.45% saline and guanidine hydrochloride. Using synthetic RNA, and decreasing the concentration of primers by five-fold and probes by 2.5-fold, changed the assay limit of detection (LOD) from 7.2 copies/reaction to 23.7 copies/reaction and the subsequent reducing of thermocycling times changed the assay LOD from 23.7 copies/reaction to 44.2 copies/reaction. However, using real clinical samples with Cq values ranging from ~12.15 to ~36.46, the results of the three tested conditions were almost identical. These alterations will not affect the vast majority of diagnostics and increase the daily testing capability in 30% and increase primers and probe stocks in 500% and 250%, respectively. Taken together, the alternative protocols described here overcome the short supply of tubes, reagents and equipment during the SARS-CoV-2 pandemic, avoiding the collapse of test offering for the population: 105,757 samples were processed, and 25,156 SARS-CoV-2 diagnostics were performed from 9 May 2020 to 30 June 2020.

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Inactivation analysis of SARS-CoV-2 by specimen transport media, nucleic acid extraction reagents, detergents and fixatives

Cited by 23 publications

References 32 publications

Performance Evaluation of the SAMBA II SARS-CoV-2 Test for Point-of-Care Detection of SARS-CoV-2

Performance Evaluation of the SAMBA II SARS-CoV-2 Test for Point-of-Care Detection of SARS-CoV-2

Analytical Sensitivity and Specificity of Two RT-qPCR Protocols for SARS-CoV-2 Detection Performed in an Automated Workflow

Overcoming Supply Shortage for SARS-CoV-2 Detection by RT-qPCR

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