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This kit has been developed for the detection of the new type of coronavirus (SARS-CoV-2) in RNA samples obtained from patients. The reaction time is approximately 1 hour, it may differ slightly depending on the qPCR device used.Within the kit, the primers and probes called N1 and N2 specifically identify the nucleocapsid (N) gene of the SARS-CoV-2 virus. These primers and probes do not recognize the SARS-CoV virus, whose genetic structure is highly similar to SARS-CoV-2. RNAseP primers and probes included in the kit recognize the human RNAseP gene and they are used to check the quality of cDNA samples prepared from patient material.The “Template DNA” contained in the kit is a synthetic DNA prepared to be used as the positive control. It contains the target regions of the N1, N2 and RNAseP genes that are amplified with the primer-probe sets included in this kit. It is not isolated from the virus, it does not have the entire virus genome and has no infectious properties.STORAGE CONDITIONSLyophilized products should be stored at -20C / +4C. Primer/probe solutions can be stored for one month at +4C. For long term storage, they should be aliquoted and stored at -20C.The primer/probe mixtures are light-sensitive and stored in brown vials. Aliquoted probes also need to be protected from light.PREPARATION OF PRIMER AND PROBE SOLUTIONSReconstitute the primer/probe mixtures with 200 ?L of nuclease-free water/TE (Tris-EDTA) buffer solution. DISSOLVING THE POSITIVE CONTROL DNADilute the Template DNA with 1000 ?L (1 mL) of nuclease-free water. Mix by pipetting without vortexing. The Template DNA solution should be aliquoted and stored at -20 C for long term storage. For short term storage, Template DNA solutions can be stored at +4C for one month.qPCR PROTOCOLNOTE: All work should be carried out on ice/cold plate.NOTE: All necessary biosecurity measures should be taken when working with patient samples.This kit is not a multiplex PCR kit; it is designed to work with 3 different qPCR reactions to be performed simultaneously in different tubes for each patient sample. The reactions (rxn) to be studied for each patient sample are shown in Table 2. All probes have been synthesized as FAM/BHQ1 labeled.Table 2. qPCR reactions to be run in separate tubes/wells for each patient sample.Primer/Probe SetTarget RegionRxn 1N1SARS-CoV-2 specific nucleocapsid geneRxn 2N2SARS-CoV-2 specific nucleocapsid geneRxn 3RNAse PHuman RNAse P geneFor each study, a negative control (NC) and a positive control (PC) reaction should be used:For negative controls, nuclease-free water should be added to the wells/tubes instead of the patient sample. The purpose of using a negative control is to determine whether there is nucleic acid contamination in any of the materials used for the amplification. A negative control reaction is mandatory in order to avoid false-positive evaluation in patient samples. No amplification should occur in negative control samples unless there is DNA contamination in any of the test materials.For positive controls, Template DNA should be added to the wells/tubes. The purpose of using a positive control is to prevent false-negative cases that may arise from study errors. In order to avoid false-negative evaluations in patient samples, positive control must be studied. Amplification should be observed in positive control samples to ensure that the reaction worked well.NOTE: The protocol shown below is the optimized protocol for the BioRad CFX Connect (1855201) qPCR device. If you use a different device, a preliminary trial should be performed using only negative control and positive control, and the reaction conditions should be optimized in accordance with the recommendations of the manufacturers of the kit and the device used, when necessary.Design the wells of the plate/strip tubes to work with 3 sets of primers/probes for each sample, and enter this design to the software of the qPCR device. An example plate design is shown below (S1: sample 1, S2: sample 2…, NC: negative control, PC: positive control)123456789101112AS1-N1S2-N1S3-N1S4-N1S5-N1S6-N1S7-N1S8-N1S9-N1S10-N1NC-N1PC-N1BS1-N2S2-N2S3-N2S4-N2S5-N2S6-N2S7-N2S8-N2S9-N2S10-N2NC-N2PC-N2CS1-RPS2-RPS3-RPS4-RPS5-RPS6-RPS7-RPS8-RPS9-RPS10-RPNC-RPPC-RPDEFGHCalculate the amount of solutions to be added to the reaction mixture for each primer-probe set (N1, N2, RNAseP) depending on the qPCR kit you use. 1 ?L of Primer/Probe mix should be added per 20 ?L reaction. Add nuclease-free water to negative control (NC) wells instead of RNA sample. Add 5 ?L of the Template DNA solution per 20 ?L reaction to the PC wells instead of the RNA sample. Cover your tube/plate with optically permeable material (cap/seal).If you are using strip tubes, spin them down. If you are using a 96 well plate, centrifuge the plate shortly (using plate rotor – for 30 sec at 500 g).Create the reaction protocol in the software of the qPCR device with the following steps: 45C-10 min95C-10 min89535051328009582915016535 cycles0035 cycles95C-5 sec60C-45 secPlace the strip tube / plate into the qPCR device and start the reaction.EVALUATION OF THE RESULTSFor each study, negative results should be obtained from NCs and positive results from PCs. Studies that do not comply with these results must be repeated.The reaction curves that are not sigmoidal should be interpreted as negative. Positive results should be obtained for RNAseP reactions with an amplification curve or cycle threshold value at or below 35 cycles. Amplification curves greater than 35 cycles for RNAseP are in the uncertainty zone. The presence of a curve, with a Cq at or below 35 cycles, for a sample in the RNAseP, indicates a positive result. Not getting a positive result for RNAseP reaction of a patient sample means that there is a problem in the RNA isolation stage. Thus, RNA isolation from those samples should be repeated.For N1 and N2 reactions, a positive result means an amplification curve or cycle threshold value at or below 35 cycles. Amplification curves greater than 35 cycles for N1 or N2 are in the uncertainty zone. The presence of a curve, with a Cq at or below 35 cycles, for a sample in the N1 or N2, indicates a positive result.As stated in Table 3, samples with positive results from all N1, N2 and RNAseP sets should be evaluated as SARS-CoV-2 positive; while N1 and N2 negative and RNAseP positive results should be evaluated as SARS-CoV-2 negative. Samples with a positive result from only one of N1 or N2 should be retested.Table 3. Evaluation of the qPCR results. N1N2RNAsePAssessment+++The patient is SARS-CoV-2 positive.--+The patient is SARS-CoV-2 negative.-++The result is uncertain.The test should be repeated.+-+ ................
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