Direct RT-qPCR Assay for the Detection of SARS-CoV-2 in Saliva Samples

Abstract

Since mid-2020 there have been complexities and difficulties in the standardisation and administration of nasopharyngeal swabs. Coupled with the variable and/or poor accuracy of lateral flow devices, this has led to increased societal ‘testing fatigue’ and reduced confidence in test results. Consequently, asymptomatic individuals have developed reluctance towards repeat testing, which remains the best way to monitor COVID-19 cases in the wider population. On the other hand, saliva-based PCR, a non-invasive, highly sensitive, and accurate test suitable for everyone, is gaining momentum as a straightforward and reliable means of detecting SARS-CoV-2 in symptomatic and asymptomatic individuals. Here, we provide an itemised list of the equipment and reagents involved in the process of sample submission, inactivation and analysis, as well as a detailed description of how each of these steps is performed.

Performance Evaluation of A Non-Invasive One-Step Multiplex RT-Qpcr Assay for Detection of SARS-Cov-2 Direct from Human Saliva

“…For instance, the Quantabio ToughMix PCR mastermix is optimised for use with crude biological samples and resistant to potential PCR inhibitors associated with such samples. We have submitted our detailed methods and protocols in a separate publication [21] for any who wish to reproduce what we are doing.…”

“…The validation work was performed with consideration to the Public Health England's (PHE) published quality guidance on the UK Standards for Microbiology Investigations (UK SMI) [20] , the Medicines and Healthcare Regulatory Agency (MHRA) target product pro le (TPP) speci cations for SARS-CoV-2 viral detection testing, and adhering to the applicable criteria de ned within the European Standard for the competence of testing and calibration laboratories (BS EN ISO/IEC 17025:2017). Whilst a parallel submitted manuscript (Tarantini et al, 2022 [21] ) provides stepwise protocols for the UoNATS assay, the article presented below de nes the quality metrics used to validate and accredit the UoNATS SARS-CoV-2 detection method, as assessed by the following parameters: Linearity and range, analytical sensitivity (limit of detection, LOD), analytical speci city, diagnostic (clinical) sensitivity and speci city, positive and negative predictive value, repeatability and sample stability, accuracy and precision, multiplex validation, robustness and reproducibility.…”

A Qualitative Evaluation of the Barriers and Enablers for Implementation of an Asymptomatic SARS-CoV-2 Testing Service at the University of Nottingham: A Multi-Site Higher Education Setting in England

“…Details of the ATS delivery and timeline, within the changing pandemic context, are provided in Figure 1 . The methods and validity of the testing approach have been published elsewhere [ 34 , 35 , 36 , 37 ].…”

Performance evaluation of a non-invasive one-step multiplex RT-qPCR assay for detection of SARS-CoV-2 direct from human saliva

“…Detailed methods have been provided in a separate publication [21] . Clinical samples are inactivated prior to downstream processing; vials are heat-inactivated in a laboratory oven with the use of a temperature probe to ensure samples are held at 95 °C for 5 minutes, shown to inactivate SARS-CoV-2 [22] .…”

“…The validation work was performed with consideration to the Public Health England’s (PHE) published quality guidance on the UK Standards for Microbiology Investigations (UK SMI) [20] , the Medicines and Healthcare Regulatory Agency (MHRA) target product profile (TPP) specifications for SARS-CoV-2 viral detection testing, and adhering to the applicable criteria defined within the European Standard for the competence of testing and calibration laboratories (BS EN ISO/IEC 17025:2017). Whilst a parallel submitted manuscript (Tarantini et al ., 2022 [21] ) provides stepwise protocols for the UoNATS assay, the article presented below defines the quality metrics used to validate and accredit the UoNATS SARS-CoV-2 detection method, as assessed by the following parameters: Linearity and range, analytical sensitivity (limit of detection, LOD), analytical specificity, diagnostic (clinical) sensitivity and specificity, positive and negative predictive value, repeatability and sample stability, accuracy and precision, multiplex validation, robustness and reproducibility.…”