INOGene-SCoV-2 RT-qPCR Detection Kit
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Instructions For Use (For Professional Use Only)

Principle and Additional Information 2.1 Specimen Collection and Pre-qPCR 2.3 TaqMan RT-qPCR Assay Storage Conditions RT-qPCR Protocol RT-qPCR program settings Analysis of Results and Interpretation 8.3 Potential Problems and Their Solutions

1. Purpose of Use INOGene-SCOV2 qPCR Detection Kit includes M-MLV Reverse Transcriptase, Taq Polymerase enzymes, Ribonuclease inhibitor, and oligonucleotides specifically designed for this kit, targeting the areas with the lowest mutation risk, (This enables the detection of all variants). It detects SARS-COV-2 viral RNA in qPCR with high sensitivity even in small quantities of RNA. This kit allows for examination of materials acquired by RNA extraction from nasopharyngeal and oropharyngeal swabs, saliva, and sputum. WHO recommends upper respiratory tract samples such as nasopharyngeal...

2.2 RT-qPCR Assay Polymerase chain reaction (PCR) is a highly sensitive and specific method used for the amplification and detection of deoxyribonucleic acid (DNA). Its conceptual simplicity has made it the most widely used technique in molecular biology and can, in theory, detect even a single copy of DNA. Therefore, it is widely used as a diagnostic test for a wide variety of bacterial, fungal, viral and parasitic pathogens. However, the genome of coronaviruses consists of ribonucleic acid (RNA) rather than DNA. Although RNA is similar to DNA, the result is unsatisfactory as Taq...

2.3 TaqMan RT-qPCR Assay Real-time PCR using 5′ nuclease or hydrolysis probes, also known as TaqMan qPCR, is an important and powerful tool used in various fields of life science. It also has great potential in diagnostic microbiology where TaqMan qPCR is often the first-line screening method for the detection of many viral or bacterial pathogens in human, animal or plant samples. TaqMan qPCR uses a pair of primers and a non-extendable probe. The probe is a short, sequence-specific oligonucleotide that binds within the region bounded by the primers. One end of the probe, usually the 5' end,...

The kit uses reporter fluorophores to target the E and RdRp genes as SARS-COV-2 controls, as well as the Rnase P gene as an internal control, as indicated in Table 2. Table 2 Targeted genes and reporter fluorophores TARGET GENE RdRp Gene Internal Control (Rnase P) 4. Storage Conditions ✔ The kit is shipped to the user with dry ice. ✔ All elements of the kit should be stored between -15 and -25 ℃. Aliquoting is recommended if thawing is to be done very frequently. ✔ The expiry date of the product is indicated on the boxes and tubes. As long as it is stored in suitable conditions, it can be...

Table 3 Quantities of Reagents that will be used in reaction Reagents are required for a reaction Reaction Mix (Taq Polymerase, RT, RI, buffer) 10 ul Primer-Probe Mix (Primer and probes of E, RDRP ve 3 ul Rnase P genes) Nuclease-free water 3 ul Total volume 16 ul 5. 16 µl of the master mix is distributed to the previously prepared PCR tubes or 96well plates. 6. Patient samples are loaded into the wells with 4 µl of the negative and/or positive control. *A negative control must be included in each analysis for contamination research. Also, when the accuracy of the results is in doubt, enzyme...

8. Analysis of Results and Interpretation 8.1 Threshold Setting When setting the threshold value, after adding an NTC well in each study and adjusting the threshold value for Rnase P to not give Ct in NTC, Rnase P Ct values are checked in patient samples, if the results are positive, RNA extraction is considered successful and Cts are checked for other genes. If patient samples do not test positive for Rnase P, the study may be contaminated or the RNA extraction was not successful. The test is retried with a new NTC. Ct values considered positive for genes are as in the Table 5. Table 5...

8.3 Potential Problems and Their Solutions Table 7 includes problems that may occur during the analysis and the solutions for them. Table 7 Potential problems and their solutions Possible Causes Suggested Solutions Incorrect setting of the Rt-qPCR The program is checked and the test is program in the software. repeated. The program is checked and the test is repeated. Wrong choice of paint or well. Reagents may not have been added correctly. (Not adding kit elements to the well, etc.) Expiration date of the kit, storage in the wrong conditions or decrease in enzyme activity after too much...

Kit optimizations were carried out using customized plasmids containing SARS-COV2 genes. Depending on the number of copies in the positive sample, the test results were evaluated and the results are shown in Table 8. Table 8 RT-QPCR analyzes with positive controls containing different number of copies Analysis Results Sample 1 Sample 2 Sample 3 Sample 4 • Kit was compared to another commercially available kit and the results are shown in Table 9. Table 9 Comparison Results Commercially Available Kit Positive 78 Positive INOGene-SCoV-2 RT-qPCR Detection Kit The possibility of false positives...

Table 10 Results of In silico analysis Middle east respiratory syndrome (taxid:1335626) Human respiratory syncytial virus (taxid:11250) Epstein barr virus (taxid:10376) E gene No match. No match. No match. No match. No match. No match. No match. No match. No match. No match. No match. No match. No match. No match. No match. No match. RdRp gene No match. No match. No match. No match. No match. No match. No match. No match. No match. No match. No match. No match. No match. No match. No match. No match.

[1] Harikrishnan, Pandurangan. "Saliva as a potential diagnostic specimen for COVID-19 testing." The Journal of craniofacial surgery (2020). [2] Bustin, Stephen A., and Tania Nolan. "RT-qPCR testing of SARS-CoV-2: a primer." International journal of molecular sciences 21.8 (2020): 3004. [3] Nagy, A., Vitásková, E., Černíková, L. et al. Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay. Sci Rep 7, 41392 (2017) https://doi.org/10.1038/srep41392 Reference/Catalogue number Expiration date Lot/Batch number Storage Conditions European...