VITAPCRTM PLATFORM

VITAPCRTM Technical Specifications

PERFORMANCE CHARACTERISTICS

Analytical Sensitivity (Limit of Detection)
LoD studies determine the lowest detectable concentration at which ≥ 95% (19/20) of the replicates were positive. The in vitro transcribed full length of SARS-CoV-2 RNA (N gene) is used in the LoD determination study. This RNA transcript stock is known titer (RNA copies/μL) spiked into the Sample Collection Buffer (SCB) consisting of negative clinical matrix to mimic clinical specimen. Samples perform standard operation procedure with the VitaPCRTM SARS-CoV-2 assay according to the instructions for use.

This data demonstrates that the VitaPCR^TM SARS-CoV-2 assay detects 2.73 copies/μl of SARS-CoV-2 RNA transcripts with a confidence ≥95%. This concentration therefore serves as the limit of detection.

Analysis of Specificity (Cross-Reactivity)
These organisms were tested in three replicates in this study. In order to demonstrate the device is specific to SARS-CoV-2, high level of these organism (i.e., 106 CFU/ml for bacteria and 105 TCID50/mL for viruses) was added into the VitaPCR^TM SARS-CoV-2 Assay, contains human A549 cell line as default matrix, and had been tested under the same operating procedure to see if there was any identified false-positive reaction.

In Silico Analysis of Specificity (Cross-Reactivity)
BLASTn analysis was performed with the primer and probe of the VitaPCR^TM SARS-CoV-2 Assay against all publicly available nucleic acid sequences in GenBank as of March 06, 2020. The nucleotide collection consists of GenBank, EMBL, DDBJ, PDB, and RefSeq sequences, but excludes EST, STS, GSS, WGS, TSA, patent sequences as well as phase 0, 1, and 2 HTGS sequences and sequences longer than 100Mb.The search parameters automatically adjust for short sequences and the expect threshold is 1000. The match and mismatch scores are 1 and -3 respectively. The penalty for creating and extending a gap is 5 and 2 respectively.

The forward and reverse primer sequences for detection of specific SARS-CoV-2 RNA showed high sequence homology to Bat coronaviruses. However, the probe sequence showed no sequence homology with SARS coronavirus, bat SARS-like coronavirus and other coronavirus genome (except for bat coronavirus RaTG13), or human genome. Combining primers and probe, there is no prediction of potential false positive RT-PCR results.

Detection of universal SARS-like RNA was designed for universal detection of SARS-CoV-2, human SARS coronavirus and bat SARS-like coronavirus, and no significant homologies with human genome, or other human coronaviruses other than SARS coronavirus that would predict potential false positive RT-PCR results. Thus, combining specific SARS-CoV-2 primer/probe set and universal SARS-like primer/probe set in multiplex RT-PCR, there is no prediction of potential false positive RT-PCR results.

Endogenous Interference Substances Study
To demonstrate that designated function of VitaPCR^TM SARS-CoV-2 Assay was operational while the specimen is tested in the presence of common endogenous substances. We tested the interfering substance individual, and spiked 2x LoD positive samples with interfering substance respectively, to reveal if there were unexpected results. Each test performed three replicates.

Interfering Substances: Table below for evaluation of interfering substances for the ability to generate false positive resuts

Interfering Substances: Table below for evaluation of interfering substances for the ability to generate false positive resuts

Clinical Performance
Due to the difficulty of obtaining clinical specimens from SARS-CoV-2 infected patients, performance characteristics of the VitaPCR^TM SARS-CoV-2 Assay has been evaluated using contrived clinical specimens. The in vitro transcribed full length of SARS-CoV-2 RNA (N gene) was used in this testing. This RNA transcript stock with known titer (RNA copies/μL) spiked at different concentrations was prepared for spiking 30 individual negative NP or OP swab specimens at different concentrations. They were blinded, randomized and spiked into Sample Collection Buffer (4 ml) in which individual negative NP or OP swab specimens were washed. 20 NP or OP swab specimens were spiked at 1.5x LoD, 5 at 3x LoD, and 5 at 5x LoD. Another 30 individual negative NP or OP swab specimens were left un-spiked. In brief, we spiked either NP or OP swab specimens into contrived samples with various concentration, then proceeded the next procedure with the VitaPCR^TM SARS-CoV-2 assay in this section. All 60 samples were blinded, handed and randomized to unbiased operators to analyze with the VitaPCR ^TM SARS-CoV-2 Assay to generate the Positive Percentage Agreement (PPA), Negative Percentage Agreement (NPA), and Overall Percentage Agreement (OPA) as a measurement of estimated Diagnostic Accuracy.