This kit uses highly conserved regions of SARS-CoV-2 Virus, Respiratory Syncytial Virus, Influenza A Virus and Influenza B Virus, and designs specific primer probes. The primers and probes can specifically bind to the target sequence. Under the action of reverse transcriptase and hot start Taq DNA polymerase, this segment is specifically amplified, and the fluorescent probe is hydrolyzed during amplification to generate fluorescence. The detection system contains dUTP-UDG enzyme to fully degrade potential product contamination and avoid false positive results; In addition, internal standards are set up to monitor the entire process of sample collection, transportation, nucleic acid extraction, and PCR amplification for highly conserved regions on the human genome, avoiding false negative results and ensuring the effectiveness of the entire process.
This kit can qualitatively detect the nucleic acids of SARS-CoV-2, Respiratory Syncytial Virus, Influenza A Virus, Influenza B Virus, providing information for clinical diagnosis; the detection system contains dUTP-UDG enzyme anti-contamination measures, which can avoid false positive results; At the same time, human-derived internal references are introduced to monitor the entire process of specimen collection, transportation, nucleic acid extraction, and PCR amplification to avoid false negative results; it takes only 35 minutes to complete detection and issue report.
Applicability
Suitable for nasopharyngeal swab sample.
High sensitivity
Three different batches of reagents were used to test, and the detection sensitivity was 500 copies/mL.
High specificity
There was no cross reaction with many common pathogens, such as Parainfluenza virus type 1, Parainfluenza virus type 3, Mycoplasma pneumoniae, Adenovirus type 7, Legionella pneumophila, Klebsiella pneumoniae, Bordetella pertussis, Measles virus, Haemophilus influenzae, Coxsackie A24, Coxsackie B1, Aspergillus flavus, Enterovirus 71, Enterovirus 70, Moraxella catarrhalis, Oral streptococcus, Streptococcus pneumoniae and Staphylococcus aureus.
Strong tolerance to inhibitors
The samples contain endogenous inhibitors (such as blood and mucin) and exogenous inhibitors (such as drugs commonly used to treat colds or other similar symptoms: oxymetazoline, sodium chloride, dexamethasone, triamcinolone acetonide, budesonide, mometasone, fluticasone, ribavirin, oseltamivir, azithromycin, tobramycin, beclomethasone, flunisolide, histamine hydrochloride, zanamivir, and palamivir), No significant impact was observed on the test results.
High accuracy
It can quickly detect SARS-CoV-2, Respiratory Syncytial Virus, Influenza A Virus, Influenza B Virus. Reference material testing results: positive coincidence rate of 100%, negative coincidence rate of 100%.
Easy to operate
PCR amplification and detection done in completely closed tube to prevent aerosol contamination.
The kit is used for the auxiliarydiagnosis and epidemiological surveillance of SARS-COV-2, RSV,Flu A and FluB infection, cannot be used as the basis for the diagnosis or exclusion of cases alone.
1. The kit should be stored at -25°C ~ -15°C away from light and avoid repeated freeze-thaw.
2. The kit can be stored for 3 days at 2-8°C after opening.
3. The kit can be stored for up to 12 months if all components are kept in the manner above. Do not use after the stated expiry date.
4. The kit can be transported in foam box sealed with ice bags or dry ice under 8°C.
Q1: The amplification efficiency is low, and there are no amplification bands in the experimental group.
A1: (1) Primers
Check if the artificially synthesized primers have degraded due to improper storage conditions; Whether the primer design is reasonable, BLAST can be used to check primer specificity or redesign primers.
(2) Template
Long term storage and repeated freezing and thawing can lead to template breakage, ring opening, or degradation. Freshly prepared DNA double strands should be used as templates; If the template GC content is too high, it will cause the double stranded DNA to be unable to open. At this time, adding PCR Enhancer (item number P021) can effectively reduce the unwinding temperature; If the template is cDNA, it is necessary to confirm the purity and integrity of the RNA used for reverse transcription.
(3) Enzymes
The enzymes used in the reaction are inactivated due to improper storage or transportation. It is recommended to replace them with new enzymes or re experiment with enzymes from another source.
(4) Amplification system
The reaction system configuration is incorrect, it is recommended to repeat the experiment.
(5) Reaction program
Check if the denaturation temperature is accurate and if the PCR instrument indicates a temperature that matches the actual temperature. If the temperature is too high, the enzyme will quickly become inactive in the first few cycles, while if the temperature is too low, the template denaturation will not be complete; The annealing temperature is not suitable. A gradient can be set for the annealing temperature to explore a suitable annealing temperature; Set annealing temperature based on primer Tm value, such as primer Tm value≥ At 72 ℃, the annealing step can be removed and the subsequent extension step (two-step PCR) can be carried out directly.
Q2: The brightness of the amplified bands is not very bright.
A2: (1) Primers
Check if the artificially synthesized primers have degraded.
(2) Template
Firstly, confirm the quality of the template. Long term storage and repeated freeze-thaw cycles can cause template breakage, ring opening, or degradation. Freshly prepared DNA double strands should be used as the template; If the template is not available, the first amplification product can be diluted in multiples and used as a template for secondary amplification.
(3) Reaction program
You can try using the Touch Down program; Extend the extension time and increase the number of cycles.
