Getting ahead of the COVID-19 pandemic entails rigorous research that can evolve as quickly as the virus. To accelerate this research, we need high-throughput assays for detecting SARS‑CoV‑2 from a large number of samples with precision, reproducibility, and speed. Other critical factors for reliable SARS-CoV-2 detection include minimal false positives and negatives, accurate capturing of transcripts down to single-digit copy numbers, reduced hands-on time, and maximizing the number of samples processed per day.
Reduce hands-on time—performing the assay on our automated platform only requires ~30 minutes of hands-on time
Process more samples—each run of 384 samples can complete in <4 hours, allowing time to run 768 samples, with up to 4 replicates, per day
Lower operating costs—our chips utilize 100-nl reactions, lowering costs for master mixes and other reagents up to 200X
Customize panels for combined pathogen tests—flexibility to vary the number of assays and samples per chip and to customize panels for combined pathogen tests (Figure 1)
Examples of molecular assays
Assays
Samples
High-throughput SARS-CoV-2 detection 768 different samples
Highly parallel ARG detection 372 different targets*
Large panels
12
*Stedtfeld, R. D. et al. Primer set 2.0 for highly parallel qPCR array targeting antibiotic resistance genes and mobile genetic elements. FEMS Microbiol. Eco.94, 2018.
Figure 1. Schematic diagram showing the flexibility of the SmartChip Real-Time PCR System. The 5,184 reaction nanowell chip allows users to set up low- to high-throughput assay panels for up to 768 samples.
Assay workflow
The SmartChip SARS‑CoV‑2 assay follows a simple workflow with ~30 min of hands-on time and ~4 hours of total processing time per 384 samples. The oligonucleotide primer and probe sequences used in this assay are identical to the primers targeting the viral nucleoprotein (N) gene used in the CDC’s FDA-approved assay for detection of SARS‑CoV‑2. The assay specifically detects the SARS‑CoV‑2 nucleoprotein genes (N1 and N2).
RNA isolated and purified from upper respiratory samples are dispensed into 5,184-well chips along with the assay mix, reverse transcribed to cDNA, and amplified using the SmartChip Real-Time PCR Cycler (Figure 2).
Figure 2. The SmartChip Real‑Time RT-PCR SARS-CoV-2 assay workflow. The SmartChip system allows you to go from sample to data in under four hours for 384 samples (with the ability to process up to 768 samples per day). Add your samples and assays to the SmartChip MyDesign Chip and then process using the SmartChip system.
Results
Assay sensitivity
The assay sensitivity was measured by determining the limit of detection (LoD), the lowest detectable concentration of SARS‑CoV‑2 RNA at which approximately 95% of all (true positive) replicates test positive. Using a limiting dilution of characterized, heat-inactivated SARS‑CoV‑2 virus of known concentration, the initial LoD was estimated to be 16 copies/µl (Table 1).
Positivity rate
Dilution
N1
N2
RP
No. of replicates
0 copies/µl
0/3
0/3
3/3
3
2 copies/µl
0/3
0/3
3/3
3
4 copies/µl
0/3
0/3
3/3
3
8 copies/µl
3/3
0/3
3/3
3
16 copies/µl
3/3
3/3
3/3
3
32 copies/µl
3/3
3/3
3/3
3
64 copies/µl
3/3
3/3
3/3
3
128 copies/µl
3/3
3/3
3/3
3
Table 1. Limiting dilution assay to determine sensitivity. The initial LoD for the SmartChip Real-Time RT-PCR SARS‑CoV‑2 assay was estimated to be 16 copies/µl. Positive and negative calls were made based on the criteria explained in the Methods Section below.
The initial LoDs were confirmed using 20 extracted replicates of the inactivated SARS‑CoV‑2 virus at the presumed LoD (16 copies/µl) and 20 replicates at 2-fold below the presumed LoD (8 copies/µl). For each replicate, a positive result for N1 and N2 was called if at least 4/5 wells showed amplification curves (<Ct 33.5 for both N1 and N2 assays); for RP, the cutoff was 1/2 wells (<Ct 33.5). Based on the result for the 20 replicates, the LoD was confirmed at 16 copies/µl—the lowest concentration where ≥95% (≥19/20) of the replicates were positive.
Positivity rate
8 copies/µl
16 copies/µl
No. of replicates
N1
N2
RP
N1
N2
RP
20
6/20
5/20
20/20
20/20
20/20
20/20
Table 2. Confirmation of LoD for the SmartChip Real-Time RT-PCR SARS‑CoV‑2 assay.
Cross-reactivity and reactivity (inclusivity)
The analytical specificity of the SmartChip Real‑Time RT‑PCR SARS‑CoV‑2 assay was demonstrated in silico following the instructions for the 2019-nCoV Kit from the CDC. The analysis evaluated the primer and probe homology with 43 organisms and viruses listed in the FDA-recommended list. Based on this analysis, a significant amplification of non-target sequences that could result in cross-reaction (false-positive results) or interference (false-negative results) was considered unlikely to occur.
The SmartChip Real‑Time RT‑PCR SARS‑CoV‑2 assay uses primers and probes identical to those recommended by the CDC that target specific genomic regions of the SARS‑CoV‑2 N gene (N1 and N2), unique to SARS‑CoV‑2. Inclusivity was determined by mapping the primers and probes to all SARS‑CoV‑2 genomes available and demonstrated to be acceptable per the amended CDC EUA (June 20, 2020).
Performance of the SmartChip Real-Time RT-PCR SARS‑CoV‑2 assay for viral detection
Experiments designed to verify the SmartChip Real‑Time RT‑PCR SARS‑CoV‑2 assay performance are described below.
Sixty pre-screened nasopharyngeal samples from individuals showing symptoms consistent with COVID-19 were acquired from a third party, 30 of which were confirmed positive, and 30 confirmed negative for the presence of SARS‑CoV‑2 using a commercial COVID-19 test (EUA202821). Technologists at Takara Bio performing the procedures were blinded to the SARS‑CoV‑2 status of the samples. The results of Takara Bio assay and third-party studies (reference assay) are summarized in Table 3. The SmartChip Real‑Time RT‑PCR SARS‑CoV‑2 assay showed a Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) of ≥95% in this study (Table 4).
Reference assay
SmartChip Real‑Time RT‑PCR SARS‑CoV‑2 assay
Reference call
Assay call
N1
N2
RP
Total
Not detected (30)
Not detected
0/30
0/30
30/30
30
Detected (30)
Detected
29/30
29/30
30/30
30
Total
60
Controls
No-template control (NTC)
0/3
0/3
0/3
3
Positive
2/2
2/2
2/2
2
Human sample control (HSC)
0/1
0/1
1/1
1
Table 3.Performance of the SmartChip Real-Time RT-PCR SARS‑CoV‑2 assay for SARS-CoV-2 detection.A blind test was conducted using the SmartChip Real‑Time RT‑PCR SARS‑CoV‑2 assay on sixty samples pre-tested for SARS-CoV-2 by a third party (reference assay). The data showed almost 100% agreement of the SmartChip Real‑Time RT‑PCR SARS‑CoV‑2 assay with the reference assay results. Positive and negative calls were made based on the criteria explained in the Methods Section below.
Passing criteria: both the PPA and NPA must be ≥95%
Reference study
Positive
Negative
SmartChip Real‑Time RT‑PCR SARS‑CoV‑2 assay
Positive
29
0
Negative
1
30
PPA: 29/30 x 100 = 96.7%
NPA: 30/30 x 100 = 100%
Table 4. Performance testing using the SmartChip Real‑Time RT‑PCR SARS‑CoV‑2 assay. Based on the unmasked SARS‑CoV‑2 status of the samples, the PPA and NPA of the SmartChip Real‑Time RT‑PCR SARS‑CoV‑2 assay were calculated according to the FDA Guidance for Industry and FDA Staff: Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests (March 13, 2007). Briefly, the paired assay results for each sample were tabulated in a 2 x 2 table, and the PPA and NPA were calculated per the equations shown.
SmartChip Real‑Time RT‑qPCR SARS‑CoV‑2 assay had a PPA and NPA of ≥95%.
Conclusions
The SmartChip Real‑Time RT‑PCR SARS‑CoV‑2 Reagents enable a sensitive, automated assay for high-throughput detection of SARS‑CoV‑2 in nasopharyngeal samples:
Allows processing of up to 786 samples per day
Low, nanoliter reaction volume eliminates the need for preamplification and saves money
Quick turnaround time <4 hrs with ~30 min hands-on-time per 384 samples
High-sensitivity (LoD: 16 copies/µl) provides increased confidence in results
Outstanding performance with PPA and NPA ≥95% in comparator studies
Methods
Primers and probes
The N1, N2, and RP primers and probes used in the assay were ordered from Integrated DNA Technologies (Cat. # 10006821–10006829). The primer sequences were identical to the N1 and N2 primers/probe sequences used in the FDA authorized original CDC 2019-nCoV real-time RT-PCR Diagnostic Panel.
Nucleic acid extraction
RNA was extracted from the samples and controls using the NucleoMag Pathogen kit (Cat. # 744210.4). Three no-template controls (NTC), one Human Sample Control (HSC) (ATCC; Cat. # MP-32), and two positive controls were included with each specimen extraction run. HSC serves as a positive control for the RP target.
When all controls exhibit the expected performance, the criteria shown in the table below were used to interpret the sample results:
HSC shows positive growth curves for 2/2 replicates for the RP gene and <3/5 replicates for N1 and N2 genes
Positive control shows positive growth curves for N1 and N2 genes in at least 4/5 replicates, and 2/2 replicates for RP gene
NTC shows no growth curves for N1, N2, or RP genes
2019 nCoV_N1
2019 nCoV_N2
RP
Expected Ct value
Assay results
Result interpretation
+
+
±
<33.5 Ct
>=4/5 for N1 and N2 assays; >=1/2 for RP assay
Sample positive for SARS-CoV-2
–
–
+
<33.5 Ct
<4/5 for N1 and N2 assays; 2/2 for RP assay
Sample negative for SARS-CoV-2
+
+
–
<33.5 Ct
>=4/5 for N1 and N2 assays; 0/2 for RP assay
Assay failure
+
–
±
<33.5 Ct
>=4/5 for N1 <4/5 for N2; >=1/2 for RP assay
Inconclusive result
–
+
±
<33.5 Ct
<4/5 for N1 >=4/5 for N2; >=1/2 for RP assay
Inconclusive result
–
–
±
<33.5 Ct
<4/5 for N1 and N2 assays; 1/2 for RP assay
Inconclusive result
–
–
–
None detected below 33.5
0/5 for N1 and N2, 0/2 for RP
Assay failure
+ represents >=4/5 replicates with amplification curves ≤Ct 33.5 – represents <4/5 replicates with amplification curves ≤Ct 33.5 ± represents 1/2 replicates with amplification curves ≤Ct 33.5
Assay performance testing
Sixty pre-tested nasopharyngeal samples from individuals showing symptoms consistent with COVID-19 infection were acquired from a third party, 30 of which were confirmed positive and 30 confirmed negative for the presence of SARS-CoV-2 using a commercial test (EUA202821) as the reference assay. These samples were stored in VTM/UTM and then frozen before shipment to Takara Bio. Technologists performing the procedures were blinded to the SARS-CoV-2 status of the samples.
On the day of the assay, all samples were thawed and extracted using the NucleoMag Pathogen kit, per the manufacturer’s user manual. Twelve microliters of eluate of each sample were assayed using the SmartChip Real-Time RT-PCR SARS-CoV-2 assay on the Takara Bio SmartChip Real-Time PCR System to determine the presence or absence of SARS-CoV-2 RNA.
