ABSTRACT
Current bottlenecks for improving accessibility and scalability of SARS-CoV-2 testing include diagnostic assay costs, complexity, and supply chain shortages. To resolve these issues, we developed SalivaDirect, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration on August 15th, 2020. The critical component of our approach is to use saliva instead of respiratory swabs, which enables non-invasive frequent sampling and reduces the need for trained healthcare professionals during collection. Furthermore, we simplified our diagnostic test by (1) not requiring nucleic acid preservatives at sample collection, (2) replacing nucleic acid extraction with a simple proteinase K and heat treatment step, and (3) testing specimens with a dualplex quantitative reverse transcription PCR (RT-qPCR) assay. We validated SalivaDirect with reagents and instruments from multiple vendors to minimize the risk for supply chain issues. Regardless of our tested combination of reagents and instruments from different vendors, we found that SalivaDirect is highly sensitive with a limit of detection of 6-12 SARS-CoV-2 copies/L. When comparing SalivaDirect to paired nasopharyngeal swabs using the authorized ThermoFisher Scientific TaqPath COVID-19 combo kit, we found high agreement in testing outcomes (>94%). In partnership with the National Basketball Association (NBA) and Players Association, we conducted a large-scale (n = 3,779) SalivaDirect usability study and comparison to standard nasal/oral tests for asymptomatic and presymptomatic SARS-CoV-2 detection. From this cohort of healthy NBA players, staff, and contractors, we found that 99.7% of samples were valid using our saliva collection techniques and a 89.5% positive and >99.9% negative test agreement to swabs, demonstrating that saliva is a valid and noninvasive alternative to swabs for large-scale SARS-CoV-2 testing. SalivaDirect is a flexible and inexpensive ($1.21-$4.39/sample in reagent costs) option to help improve SARS-CoV-2 testing capacity. Register to become a designated laboratory to use SalivaDirect under our FDA EUA on our website: publichealth.yale.edu/salivadirect/.
ABSTRACT
The current RT-qPCR assay recommended for SARS-CoV-2 testing in the United States requires analysis of three genomic targets per sample: two viral and one host. To simplify testing and reduce the volume of required reagents, we developed a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the CDC, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the singleplex assay adapted for research purposes. Low copies (>500 copies / reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current singleplex diagnostics by saving reagents, costs, time and labor.
ABSTRACT
Rapid and accurate SARS-CoV-2 diagnostic testing is essential for controlling the ongoing COVID-19 pandemic. The current gold standard for COVID-19 diagnosis is real-time RT-PCR detection of SARS-CoV-2 from nasopharyngeal swabs. Low sensitivity, exposure risks to healthcare workers, and global shortages of swabs and personal protective equipment, however, necessitate the validation of new diagnostic approaches. Saliva is a promising candidate for SARS-CoV-2 diagnostics because (1) collection is minimally invasive and can reliably be self-administered and (2) saliva has exhibited comparable sensitivity to nasopharyngeal swabs in detection of other respiratory pathogens, including endemic human coronaviruses, in previous studies. To validate the use of saliva for SARS-CoV-2 detection, we tested nasopharyngeal and saliva samples from confirmed COVID-19 patients and self-collected samples from healthcare workers on COVID-19 wards. When we compared SARS-CoV-2 detection from patient-matched nasopharyngeal and saliva samples, we found that saliva yielded greater detection sensitivity and consistency throughout the course of infection. Furthermore, we report less variability in self-sample collection of saliva. Taken together, our findings demonstrate that saliva is a viable and more sensitive alternative to nasopharyngeal swabs and could enable at-home self-administered sample collection for accurate large-scale SARS-CoV-2 testing.
ABSTRACT
The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays are being used by clinical, research, and public health laboratories. However, it is currently unclear if results from different tests are comparable. Our goal was to evaluate the primer-probe sets used in four common diagnostic assays available on the World Health Organization (WHO) website. To facilitate this effort, we generated RNA transcripts to be used as assay standards and distributed them to other laboratories for internal validation. We then used (1) RNA transcript standards, (2) full-length SARS-CoV-2 RNA, (3) pre-COVID-19 nasopharyngeal swabs, and (4) clinical samples from COVID-19 patients to determine analytical efficiency and sensitivity of the qRT-PCR primer-probe sets. We show that all primer-probe sets can be used to detect SARS-CoV-2 at 500 virus copies per reaction, except for the RdRp-SARSr (Charite) confirmatory primer-probe set which has low sensitivity. Our findings characterize the limitations of currently used primer-probe sets and can assist other laboratories in selecting appropriate assays for the detection of SARS-CoV-2.
