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vDetect COVID-19 RT-qPCR
The vDetect COVID-19 RT-qPCR diagnostic kit is a qualitative in vitro test designed to detect the presence of SARS-CoV-2 coronavirus genetic material in samples of biological material obtained primarily from the human upper airways (nose and nasopharynx) using the RT-qPCR method. The kit is intended exclusively for use in a diagnostic laboratory with the appropriate equipment, safety standards and properly trained personnel.
The diagnostic kit vDetect COVID-19 RT-qPCR is based on improved protocol developed by Prof. Dorsten in Charité Berlin, Germany referenced by WHO. The RT and qPCR are carried out in one tube in 1-step fashion. The kit contains 2 sets of primers and TaqMan probe. One set is for the detection of gene E and the second set is for the detection of gene RdRP of SARS-CoV-2 virus. Both probes are labeled with FAM for gene E and RdRP gene.The proposed diagnostic algorithm is to carry out the E gene assay first as the screening test and subsequently in case of positive or ambiguous results to carry out the RdRP assay as the confirmatory test. But advanced performance of RdRP allows essay design exchange. The kit contains the full genomic RNA of SARS-CoV-2 virus as a positive control material. The qPCR master mix of the kit contains ROX passive reference dye for fluorescence normalization between wells (can be modified to no ROX/low ROX/high ROX upon customer request). Onepackage of the kit is sufficient for 500 reactions.
1Clinical performance of the vDetect COVID-19 RT-qPCR kit was performed for the E gene and for the RdRP gene. The evaluation was performed on a selected set of 38 positive and 54 negative clinical samples of patients, which were confirmed by a reference method used for routine testing by regional public health authorities of the Slovak Republic (original WHO protocol). Testing of this selected set of samples was performed independently in two laboratories; one of the laboratories was working with blinded samples. Analyzes in both laboratories clearly confirmed the results of the reference method for all samples evaluated and demonstrated the high reliability and reproducibility of the results obtained with the vDetect COVID-19 RTqPCR kit. Based on the data obtained, the vDetect COVID-19 RT-qPCR kit has 100% diagnostic sensitivity and 100% diagnostic specificity. At the same time, it is not possible to determine the proportion of falsenegative and false-positive results.
2Evaluation of analytical sensitivity (LOD – limit of detection) was performed for the E gene and for the RdRP gene. The test was performed using the control material "SARS-CoV-2 Standard" (Exact Diagnostics), which in the undiluted state contains 200 copies of the template per 1 μl. Dilutions with a concentration of 40 copies/μl (= 200 copies/reaction), 8 copies/μl (= 40 copies/reaction), 1.6 copies/μl (= 8 copies/reaction were used in the analytical sensitivity test. A dilution medium prepared by mixing extracted RNA from samples taken from several COVID-19 suspect patients with a negative RT-qPCR diagnostic test result was used to dilute the control material. The dilution medium was used to mimic as closely as possible the characteristics of the standard clinical samples as a complex mixture, including the possible content of RT-qPCR inhibitors. The assay was performed in 8 replicates for each dilution prepared. The test confirmed the high sensitivity of the vDetect COVID-19 RT-qPCR diagnostic kit. Reliable template detection, to determine the presence of both the E gene and the RdRP gene, was demonstrated up to a dilution containing 8 copies per reaction (1.6 copies per 1 μl sample). A positive signal was observed in some, but not all, 1.6 and 0.32 copy reactions. Thus, the reliability of template detection at these dilutions is less than 100% efficient (see table and graphs below).), 0.32 copies / μl (= 1.6 copies/reaction) and 0.064 copies/μl (= 0.32 copies/reaction), which
3Evaluation of specificity (cross-reactivity to other coronaviruses) was performed for the E gene and for the RdRP gene. The test was performed using the control material "Coronavirus RNA specificity panel" (EVAg, European Virus Archive - Global), which contains RNA viruses HCoV-229E, HCoV-OC43, HCoV-Nl63, SARS-CoV HKU39849, MERS-CoV and SARS-CoV -2, each in a separate tube. The assay was performed in 3 replicates for each of the indicated viruses.The test confirmed 100% specificity of the vDetect COVID-19 RT-qPCR diagnostic kit. A positive result was recorded exclusively in reactions containing SARS-CoV-2 RNA, as well as in assays for the presence of the E gene and the RdRP gene.
4Occurrence of contamination by synthetic positive controls in various commercially available products used to perform RT-qPCR diagnosis of COVID-19 disease, e.g. primers, probes or RT-PCR mixtures is a global problem. The mixture of primers and probes SARS-CoV-2_E in the vDetect COVID-19 RT-qPCR kit was designed not to amplify the most commonly used synthetic positive controls at all or only with low efficiency. Thus, the vDetect COVID-19 RT-qPCR kit can also be used effectively in workplaces that have a problem with contamination when testing for the presence of the SARS-Cov-2 E gene.
Evaluation of chemical stability (non-specific reaction between oligonucleotides contained in the individual primer and probe mixtures) was performed for the E gene and for the RdRP gene. The assay was performed as a multiple analysis of non-template controls (NTC) of 20 replicates for the E gene and 40 replicates for the RdRP gene. The assay confirmed the high chemical stability of the oligonucleotides contained in the vDetect COVID-19 RT-qPCR kit. In each of the analyzed NTCs, a negative result was recorded with no indication of an increase in signal or the presence of amplification.
Registration code: P1048A
ESET, spol. s r.o.
IČO: 31 333 532
Einsteinova 24, 851 01 Bratislava, Slovenská republika
+421 2 322 44 111
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