
POLGEN CORONAVIRUS DETECTION KIT
Coronaviruses are a group of viruses that cause diseases in mammals and birds. Although the pathologies they create in humans are usually in the form of mild respiratory infections, several coronavirus types cause severe pathologies such as SARS, MERS, and COVID-19. Coronavirus Detection Kit.
Coronaviruses belong to the Orthocoronavirinae subfamily of the Cornidoviridae family in Nidovirales subgroup and are single-stranded RNA viruses. They are known by the envelope structure around them and contain a positive sense RNA sequence. Their genome lengths range from about 25 to 35 kb and carry crown-like proteins of about 20 nm in size.
The SARS-CoV-2 virus, which was first seen in Wuhan in 2019, is the causative agent of COVID-19 which has spread all over the world. SARS-CoV-
2 is a coronavirus similar to SARS-CoV and COVID-19 manifests by high fever, shortness of breath and dry cough in infected patients. The mortality rate of this disease increases with age, and it is observed to be more severe in men and especially in smokers and those with chronic diseases. COVID-19 infection turns into pneumonia when it is severe.
How to take samples for this test?
The World Health Organization (WHO) recommends RT-PCR-based testing to establish a definitive diagnosis in cases where COVID-19 infection is suspected. It is recommended that the samples to be taken for this test are taken from the respiratory tract (mouth swab, nose swab, or mucus sample (if any), etc.).
How should the samples be transferred?
After taking the samples, it is very important to keep them in the cold chain. In the cases where the test laboratories can be reached immediately, the samples can be kept at +4 °C, in cases where it will take a long time to reach the test centers, the samples should be frozen at –20 °C or preferably at -70 °C and kept in dry ice.
What types of tests can be done for the diagnosis of coronavirus?
In order to conduct molecular tests, the relevant laboratories must meet the biosecurity requirements at least at the BSL-2 level. After the swabs arrive in the laboratory, RNA should be isolated from the samples. It is not recommended to use heat-treatment prior to RNA isolation. WHO has published qRT-PCR methods developed by China, Germany, Hong Kong, Japan, Thailand, USA, and France on its website and stated that one of them can be used for SARS-CoV-2 diagnosis. One of the most reliable of these methods is the method developed by the CDC agency of the USA; however, in the validation studies, this test method was observed to sometimes give false-positive results.
What is POLGEN Coronavirus qRT-PCR kit?
Coronavirus qRT-PCR test kit, developed by POLGEN Biotechnology A.Ş. has the ability to give results within 1 hour after RNA isolation. It is specially designed to diagnose SARS-CoV-2 virus, which causes COVID-19 disease. Our kit eliminates the problems (primary-dimer, probe-dimer formation) associated with the kit developed by CDC, and is currently the most accurate kit on the market. It contains primer-probe sets that recognize 3 different regions in the N gene of the SARS-CoV-2 virus in addition to the positive control primer-probe set and the positive control DNA sample.
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SARS-CoV-2(COVID-19) qRT-PCR Detection Kit User Guide
- This kit has been developed for the detection of the new type of coronavirus (SARS-CoV-2) in cDNA samples obtained from patients. After RNA is isolated from patient samples, it should first be converted to cDNA by standard
- The reaction time is 1 hour, it may differ slightly depending on the qPCR device
- 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
- 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
STORAGE CONDITIONS
- Undiluted products should be stored at -20 °C / +4 °
- Solutions of primers (10 µM) and probes (5 µM) and template DNA can be stored for one month at +4 °
- The probes are light-sensitive and stored in brown vials. Aliquoted probes also need to be protected from
PREPARATION PRIMER AND PROBE SOLUTIONS
Reconstitute the primers and probes with 200 µL of nuclease-free water/TE (Tris-EDTA) buffer solution.
DISSOLVING THE POSITIVE CONTROL DNA
- Dilute the Template DNA with 100 µL of nuclease-free water. Mix by pipetting without vortexing. The resulting Template DNA solution is a stock solution; it should be aliquoted and stored at -20 °
- To prepare the Template DNA solution at working concentration, dilute the stock DNA solution by 1:10. For example, 10 µL Template DNA Stock Solution + 90 µL nuclease-free water should be used for preparing 100 µL of Template DNA Working Solution. Template DNA Working Solutions can be stored at + 4 °C for one
qPCR PROTOCOL
NOTE: 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
Table 2. qPCR reactions to be run in separate tubes/wells for each patient sample.
Primer/Probe Set | Target Region | |
Rxn 1 | N1 | SARS-CoV-2 specific nucleocapsid gene |
Rxn 2 | N2 | SARS-CoV-2 specific nucleocapsid gene |
Rxn 3 | RNAse P | Human RNAse P gene |
- For 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 DNA contamination in any of the materials used for the 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
NOTE: The protocol shown below is the optimized protocol for the BioRad CFX Connect (1855201) qPCR device using iTaq Universal Probes Supermix (BioRad, 1725130). If you use a different kit/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.
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- 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)
- Calculate the amount of solutions to be added to the reaction mixture for each primer-probe set (N1, N2, RNAseP) according to Table
Table 3. Reaction mixtures to be prepared separately for each primer-probe set (N1, N2, RNAseP). N = n + 1 if the number of samples to be run at once (including the controls)
(n) is 14 or less, and N = n + 2 if it is 15 or more.
Amount to be added for a 20 µL reaction | |
iTaq Universal Probes
Supermix (2x) |
N x 10 µL |
10 µM F primer | N x 1 µL |
10 µM R primer | N x 1 µL |
5 µM probe | N x 1 µL |
Nuclease-free water | N x 2 µL |
- Prepare the mixes for each set according to Table 3 and mix them by pipetting and spin them
- Add 15 µL of the mix of a specific set to the designated wells of that specific
- After the solutions are aliquoted to the wells, add 5 µL of the cDNA samples to the respective
- Add 5 µL of nuclease-free water to negative control (NC) wells instead of
- Add 5 µL of the working solution for Template DNA to the PC
- 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:
95 °C-5 min
95 °C-5 sec 40 cycles
60 °C-30 sec
- Place the strip tube / plate into the qPCR device and start the
EVALUATION OF THE RESULTS
- For each study, negative results should be obtained from NCs and positive results from Studies that do not comply with these results must be repeated.
- Positive results should be obtained for RNAseP reactions for each Not getting a positive result for RNAseP reaction of a patient sample means that there is a problem in the RNA isolation/cDNA extraction stages. Thus, RNA isolation and cDNA production from those samples should be repeated.
- As stated in Table 4, 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
Table 4. Evaluation of the qPCR results.
N1 | N2 | RNAseP | Assessment |
+ | + | + | 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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