This kit uses fluorescent PCR to design primers and probes targeting conserved regions of enterovirus 5 UTR and VP1 genes. The 5 ' of the probe is labeled with the reporting group, and the 3' is labeled with the quenched group. In the processof PCR amplification, when the probe kept the complete sequence, the fluorescence of the group was reported to be absorbed by the quenched group without producing fluorescence; when the probe was combined with the template. the probe would be cut off by Taq enzyme (5 '-3' exonuclease activity); after thegroup was separated from the quenched group, the fluorescence could not be absorbed by the quenched group and produced fluorescence signal. The fluorescence PCR instrument will automatically draw a fluorescence curve according to the detected fluorescence signal and determine the detection result. With human P globin gene as internal reference (IC) gene, this kit can quality control the whole extraction and detection process.
� High sensitivity
The kit can detect Echovirus with detection rate equal or higher than 95% at the concentration equal or higher than 500 copies/mL.
� Accurate diagnosis
The kit is used for qualitative detection of enterovirus universal (EVU, include A2, A4, A5, A6, A7, A9, A10, A12, A16, B1, B2, B3, B4, B5, enterovirus 71 and Echovirus, etc.), and can distinguish enterovirus 71(EV71), Coxsackievirus A6 (CA6), Coxsackievirus A10 (CA10) and Coxsackievirus A16 (CA16).
No cross reactivity with Norovirus, Herpes simplex virus 1, Herpes simplex virus 2, Enteric adenovirus, Varicella zoster virus, Rotavirus, Epstein-Barr virus, Rubella virus, Measles virus, Influenza A virus, Parainfluenza virus, Influenza B virus, Human cytomegalovirus, Respiratory syncytial virus, Group B Streptococcus, Klebsiella pneumoniae, Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus, Salmonella, Haemophilus influenzae, Mycoplasma pneumoniae, Adenovirus and Human genomic DNA , etc.
� Strong stability
Endogenous inhibitors (blood, mucin, etc.) and exogenous inhibitors (contains medications commonly used to treat or treat similar symptoms such as Dexamethasone, Ribavirin , Oseltamivir ,etc.) in the samples had no significant effect on the test results.
This kit can detect coxsackie virus A2, A4, A5, A6,A7, A9,A10, A12,A16. B1.B2. B3, B4, B5, enterovirus 71 and Echovirus, etc. The test results are only used for in vitro diagnostic aid and should not be used asthe sole basis for clinical diagnosis, treatment or management of patients.
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 7 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.
