Specimens for the initial assay evaluation
The remaining specimens collected during routine clinical care (166 nasopharyngeal swabs), which would otherwise have been discarded, were used in the evaluation of our system. Samples were collected by trained personnel using nasopharyngeal Eswab (Copan, Brescia-Italy) and processed at LifeGene srl Laboratory Messina (Italy) using a commercially available system: Novel Coronavirus Real-Time Multiplex RT-PCR Kit (2019-nCoV) (3-gene detection) (Life River, San Diego, CA, USA). The Life River system is one of those included in the list approved by the WHO16,17.
Specimens for comparison with other commercial kits
To evaluate the performance of our assay, we tested it head to head with three other commercially available kits, namely, the RIDAGENE SARS-CoV-2 test (R-Biopharm AG, Darmstadt, Germany), which is a single target gene assay (detecting the E gene); the Real-Time Fluorescent RT-PCR Kit for Detecting SARS-CoV-2, which identifies the ORF1ab gene as a domain target (BGI Genomics Co. Ltd. Yantia, China); and the Allplex SARS-CoV-2 assay, which detects four target genes: the RdRp/S and N genes specific for SARS-CoV-2 and the E gene for all sarbecoviruses, including SARS-CoV-2 (Seegene Inc., Seoul, South Korea). A total of 40 remnants of specimens collected for routine clinical care (40 nasopharyngeal swabs) were used for the test comparison.
Kit design
Our diagnostic assay is a probe-based qualitative reverse transcriptase real-time PCR (qRT-PCR) probe. The targeted COVID-19 genes detected by our assay are the RNA-dependent RNA polymerase (RdRp), envelope (E), and nucleocapsid (N) of COVID-19. The primers and probes were designed based on the published sequence of COVID-19 in NCBI (reference sequence NC 045512.2) and were synthesized by Bio-Fab Research (Bio-Fab Research, Rome, Italy). The two sets of primers were specific to COVID-19: the “E” primer for the envelope gene and the “N” primer for the nucleocapsid gene. One of them is called RdRp, which targets the polymerase gene and is common with the SARS virus. The concentrations of primers and probes were determined by experimental procedures, and the sensitivity of the test was carried out with the chimeric plasmid described below. Primer and probe sequences are not shown as the kit is protected intellectual property (MOLgen-COVID-19; SARS-CoV-2 Real Time RT-PCR kit, by Adaltis).
A portion of an endogenous human β-actin gene was used as an internal control (IC) for the test, the latter also allowing the evaluation of correct nasopharyngeal sampling.
To evaluate our kit, an initial proficiency assay was carried out in the microbiology laboratories of the Department of Experimental Medicine of the “Tor Vergata” University of Rome. This proficiency assay was run using chimeric plasmids (CPs) in which virus sequences were artificially inserted into a plasmid [pBlueScript II SK(+)]. The synthesis of CP was contracted for Bio-Fab Research (Bio-Fab Research, Rome, Italy).
The analytical specificity and cross-reactivity of the primers and probes in our kit were evaluated using ZeptoMetrix panels (ZeptoMetrix, Co., Buffalo, NY, USA): (a) NATtrol SARS-CoV-2 (E/ORF/1ab recombinant) Stock (ZeptoMetric); (b) NATtrol SARS-CoV-2 (recombinant-only N) Stock; and (c) NATtrol Coronavirus-SARS Stock. Stock is formulated with intact and purified bacterial cells that contain synthetic SARS-CoV-2 sequences (the cells have been chemically modified to render them noninfectious and stable in a refrigerator) (Table 4). The panels are supplied in a purified protein matrix that mimics the composition of real clinical samples. Cross-reactivity was evaluated using (1) the NATtrol Respiratory Verification Panel (Zeptometrix NATRVP2-BIO) containing 22 viral and bacterial targets and (2) NATtrol MERS-CoV Stock (NATMERS-ST). Both panels contained intact viral and/or bacterial particles chemically modified to render them noninfectious and stable in a refrigerator.
The analytical sensitivity or limit of detection (LoD) of the test was determined by serial dilution with the AccuPlex SARS-CoV-2 v2 Reference Material Kit containing 5175 RNA cp/mL of inactivated SARS-CoV-2 virus and the research reagent for SARS-CoV-2 RNA (NIBSC code 19/304) obtained from the National Institute for Biological Standards and Control (NIBSC, UK). We treated the samples from both panels as a common nasopharyngeal sample but in triplicate (Table 5).
Assay conditions
A 200-µl aliquot of samples collected in Eswab was extracted with a manual procedure using the magnetic silica bead procedure (MOLgen Universal Extraction Kit, QIAamp viral RNA) according to the manufacturer’s instructions. To process many samples at once, the extraction procedure was also automated at ExtraLab (Adalties Srl, Guidonia, Italy).
Real-time amplification was performed with AmpliLab (Adalties Srl) and CFX96 (Bio-Rad, Hercules, CA, USA) using qPCRBIO PROBE 1-Step Go No-Rox (PCR biosystems; www.pcrbio.com). To establish the appropriate amount of reverse transcriptase activity, RTase Go quantitation was performed according to the manufacturer’s instructions. The titration experiment showed that 0.2 µl of 20× RTase Go in the amplification mix gave good results in terms of sensitivity.
We achieved the ideal reaction condition using a 20-µl reaction master mix composed as follows: 2× qPCRBIO probe 1 step Go Mix 10 µl; ppMix (a mixture of primer and probe) 5 µl; 20X RTase Go 0.2 µl; and 5 µl of sample. In particular, ppMix contained the following final concentrations: 10 pmol of RdRp, E and β-actin (forward and reverse primers), 30 pmol of N (forward and reverse primer), and 2.5 pmol of the probe for each target.
The RT-PCR conditions for both instruments were as follows: one step at 45 °C for 10 min; a step at 95 °C for 2 min; 40 steps at 95 °C for 5 s; and the last step at 60 °C for 25 s.
The instrument was programmed to read the RdRp gene in Fam, the E gene in Rox, the N gene in Cy5, and β-actin in the Hex channel.
The kit is now distributed worldwide by Adaltis, and the commercial name is MOLgen-COVID-19 Real Time RT PCR.
Upcoming updates
The kit has recently been updated with detection of an additional gene, S (spike gene), which is read in the Cy5-5 channel. Figure 4 reports the curve and relative CT of a positive sample.
Comparison test with other commercial kits
The comparison test was performed using 40 additional nasopharyngeal swabs that were examined with three commercially available kits (above reported) following the manufacturers’ instructions (Table 6).
All methods described were carried out in accordance with relevant guidelines and regulation and the study was approved by Independent Ethics Committee Tor Vergata Polyclinic on 25 June 2020.
Ethics approval
The study did not include human participants but leftover samples. Specific informed consent are not required (as stated by “Independent Ethics Committee Tor Vergata Polyclinic on 17 June 2020, having based this study on the use of leftover human specimens collected for routine analysis that would otherwise been discarded. The same specimens are “unlinked anonymized materials”. This statement is in agreement to FDA “Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable” April 25, 2006, and “Bioetica ed uso dei campioni biologici umani” Pezzati P. & Graziani MS. Biochimica Clinica, 2008, vol. 32, n. 3.
