Primer group and probe for detecting novel coronavirus 7a subgene as well as application and product of primer group and probe
A coronavirus and primer set technology, applied in the biological field, can solve the problems of poor sensitivity, long time consumption, complicated operation, etc., and achieve the effects of good specificity, improved sensitivity, and low sensitivity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0024] Example 1 Primer and probe design
[0025] Download the whole genome sequence of the novel coronavirus from the GISAID database, use MAFFT software to perform sequence alignment, intercept the 5' UTR region Leader sequence and the 5' end of the 7a gene to form a primer probe design template, and use Primer express3.0 and Primer 5.0 software to design Fluorescent RT-RAA primers and Taqman probes. The designed upstream primer falls into the Leader region, the downstream primer falls into the 7a gene region, and the probe sequence can fall into any region or span the 2 regions. This design can amplify the 7a subgene and avoid the simultaneous amplification of the 7a gene RNA. The designed primer probes were aligned using NCBI BLAST, and the homology with SARS, MERS, 229E, OC43, NL63, HKU1 coronavirus and other microorganisms and human genomes was excluded. The 5' end of Taqman probe was labeled with FAM fluorophore, and the 3' end was labeled with BHQ1 fluorophore. The ...
Embodiment 2
[0028] The optimization of the primer of embodiment 2
[0029] In order to achieve the best amplification effect, a single base point mutation was performed on the upstream primer sequence, and the base was mutated from C to A (SEQ ID NO. 4), so that the difference between the GC content and Tm value of the upstream and downstream primers was more Small, reduce the mismatch rate and improve the amplification efficiency. Using nucleic acids extracted from COVID-19 positive clinical samples (2020-COVID-19-1 and 2020-COVID-19-2), the primer effects before and after optimization were compared ( figure 1 ), the results showed that the peak time of the primer amplification curve after point mutation was earlier, and the final fluorescence value was larger, achieving better amplification effect.
Embodiment 3
[0030] Example 3 Establishment and optimization of fluorescent RT-RAA detection method
[0031] Using Qiagen Viral RNA Mini Kit extracts 200 μL of RNA from inactivated new coronavirus cell culture medium as amplification template, and the template concentration is fixed. Fluorescence signal intensity determines optimal primers, probe concentrations and reaction conditions. The optimized fluorescent RT-RAA reaction system is: buffer IV 25 μL, upstream primer (ie SEQ ID NO. 4) 2.1 μL (10 μmol / L), downstream primer 2.1 μL (10 μmol / L), probe 0.6 μL (10 μmol / L) ), 12.7 μL of RNAase-free sterile water. The optimized amplification reaction conditions were as follows: after pre-amplification at 39°C for 7 minutes, the reaction temperature was set to 39°C, amplification was performed for 30 minutes, and the results were observed in real time.
PUM
| Property | Measurement | Unit |
|---|---|---|
| Copy number | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More