Protocol validation data
One of our goals with SalivaDirect™ is to test a number of alternate reagents, and combinations of alternate reagents, at each step.
This will add some redundancy and flexibility in the supply chain (NEB backed up on kit orders? Order from Thermo!) as well as allow labs to substitute whatever reagents they have or have access to.
Limit of detection
A positive saliva specimen from a confirmed COVID-19 patient with a known virus concentration (3.7 × 104 copies/µL) was spiked into saliva collected from health care workers who tested negative for SARS-CoV-2 using the CDC assay.
Spiked saliva specimens were treated with proteinase K, heat inactivated, and tested in the dualplex RT-qPCR assay.
Input volumes, matrices and RT-qPCR programs are the same for each combination of proteinase K, RT-qPCR kit, and RT-qPCR instrument.
The following 2-fold dilutions series of 400, 200, 100, 50, 25, 12, and 6 copies/µL were tested in triplicate to determine the preliminary limit of detections.
The final limit of detection was confirmed with 20 additional replicates.
We used this approach to determine the limit of detection for proteinase K and RT-qPCR kits from 3 different vendors, and 3 RT-qPCR instruments (See Table 1).
Stability of spiked-in saliva samples used for the limit of detection experiment was determined by placing 3 different concentrations (12, 25, and 50 copies/µL) for 7 days under different temperature regimes (4°C, room temperature, and 30°C). Results were compared to results obtained in the limit of detection experiment (fresh). Samples were treated with ThermoFisher Scientific proteinase K and tested with the ThermoFisher TaqPath RT-qPCR kit on the Bio-Rad CFX96. Overall, samples were stable under each condition for at least 7 days.
In addition to cross-reactivity tests performed by the CDC, we have tested SalivaDirect™ on 52 saliva specimens collected from adults during the 2018/2019 and 2019/2020 (pre-COVID19) autumn/winter influenza seasons and detected 51 negative, and 1 invalid results.
Performance of SalivaDirect™ was compared to the authorized ThermoFisher Scientific TaqPath RT-PCR COVID-19 kit by testing 37 paired positive and 30 paired negative nasopharyngeal (NP) and saliva samples. Nasopharyngeal swabs and saliva were collected from inpatients and healthcare workers in the Yale-New Haven Hospital. Saliva was collected in sterile urine cups or 5 mL tubes without addition of any preservatives. The ThermoFisher Scientific TaqPath COVID-19 combo kit combines RNA extraction using the MagMax Viral/Pathogen Nucleic Acid Isolation Kit with a multiplex RT-PCR diagnostic assay targeting 3 regions of the SARS-CoV-2 genome (N, S, and ORF1ab). For SalivaDirect™ we used the ThermoFisher Scientific proteinase K, ThermoFisher Scientific TaqPath RT-PCR kit, and Bio-Rad CFX96 instrument.
We selected the positive and negative pairs based on preliminary results of our modified research protocol based on the US CDC primer-probe sets. Out of the 37 nasopharyngeal swabs that were tested with the TaqPath COVID-19 kit, three specimens tested negative (Ct value cut-off of 37). However, earlier results with the modified CDC assay indicated a (weakly) positive signal, and the paired saliva specimen tested positive with both SalivaDirect™ and the TaqPath COVID-19 kit. Two saliva specimens tested negative for SalivaDirect™ while the NP swab and saliva tested positive or inconclusive with the TaqPath COVID-19 kit.
All negative NP and saliva specimens tested negative with both the TaqPath COVID-19 kit and SalivaDirect™.
Important Regulatory Information about SalivaDirect™
SalivaDirect™has not been FDA cleared or approved. It has been authorized by the FDA under an emergency use authorization for use by authorized laboratories. The test has been authorized only for the detection of nucleic acid from SARS-CoV-2, not for any other viruses or pathogens. This test is only authorized for the duration of the declaration that circumstances exist justifying the authorization of emergency use of in vitro diagnostic tests for detection and/or diagnosis of COVID-19 under Section 564(b)(1) of the Act, 21 U.S.C. § 360bbb-3(b)(1), unless the authorization is terminated or revoked sooner.