CN114085924A - Novel coronavirus 4-point mutation S gene identification kit based on high-resolution melting curve and identification method thereof - Google Patents
Novel coronavirus 4-point mutation S gene identification kit based on high-resolution melting curve and identification method thereof Download PDFInfo
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Abstract
The invention discloses a novel coronavirus 4-point mutation S gene identification kit based on a high-resolution melting curve and an identification method thereof, which relate to the field of biomedicine and are characterized in that amplification primers are designed according to 4 different mutation sites of a novel coronavirus S gene, and the S gene with different mutation sites is identified through an optimized high-resolution melting curve reaction system. The kit has the advantages of simple operation, high reaction speed, no need of post-treatment of PCR products, operability and reproducibility, and can be used for identifying novel coronavirus S genes with different site mutations.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a novel coronavirus point mutation S gene identification kit based on a high-resolution melting curve, and a detection method and application thereof.
Background
Since the outbreak of new coronary pneumonia epidemic, a huge disaster is brought to the human society, and the new coronary pneumonia epidemic is not effectively controlled yet. The rapid propagation of the novel coronavirus not only seriously affects the development of global public health service, but also puts higher requirements on port health quarantine and epidemic prevention and control. Genetic mutations are a common phenomenon in the natural evolution of viruses, and studies have shown that novel coronavirus mutants may confer either greater transmission or capacity to evade certain antibody immune responses. Spike protein (S) on the surface of the new coronavirus plays an important role in virus propagation, and rapid identification of the S gene mutant virus is not only beneficial to epidemic situation prevention and control, but also beneficial to further monitoring the propagation and evolution process of the virus. Therefore, the development of safe, rapid, accurate and sensitive mutant gene rapid detection methods is urgently needed for port new coronary quarantine so as to realize rapid identification of novel coronavirus S gene mutants.
In recent years, with the development of molecular biology techniques, single-stranded conformational isomeric polymorphism analysis (SSCP), high performance liquid chromatography, and first, second, third generation sequencing, metagenomic sequencing and other molecular means have been successfully applied to the identification of known or unknown mutant genes. However, these identification methods all have disadvantages: although the sensitivity of SSCP is higher, the normal chain and the mutant chain can be distinguished only by electrophoresis, the mutation type and the mutant gene locus still need to be further sequenced, and the requirement of the electrophoresis condition is strict; the high performance liquid chromatography can only check whether mutation exists or not, can not detect the mutation type, and the result judgment is easy to make mistakes; the sequencing technology is widely applied, but is long in time consumption and high in cost, and is not suitable for the requirement of rapid port detection. In view of the above-mentioned shortcomings, a new molecular identification means needs to be searched for safely, rapidly, accurately and sensitively performing molecular identification of a mutant gene.
Disclosure of Invention
Aiming at the defects or improvement requirements of the prior art, the invention provides a novel coronavirus point mutation S gene identification kit based on a high-resolution melting curve, which can be used as a novel identification means for a mutation S gene, identifies the mutation gene on the basis of melting curves of different forms formed by different melting temperatures of mononucleotides, has the advantages of simple operation, high speed, low cost, high flux, accurate result, no limitation of detection sites, no need of post-treatment on PCR products, capability of detecting the difference of single base and the like, realizes real closed-tube operation so as to reduce the pollution risk, and is very suitable for analysis of a large number of samples.
The invention is realized by the following technical scheme:
the invention designs a group of novel coronavirus N501Y, A570D, S982A and D1118H site mutated S gene identification primers based on a high-resolution melting curve, and the primers have strong specificity and high sensitivity. The sequence is as follows:
N501Y-F：5’-AGCACACCTTGTAATGGTGTTGAAG-3’ ；
N501Y-R：5’-TCCACAAACAGTTGCTGGTGCATGT-3’ 。
A570D-F：5’-GTTTAACAGGCACAGGTGTTCTTAC-3’ ；
A570D-R：5’-CACCACCAAAAGAACATGGTGTAAT-3’ 。
S982A-F：5’-CAAGCTTTAAACACGCTTGTTAAAC-3’ ；
S982A-R：5’-TGTCTGCAAACTTTGAAGTCTGCCT-3’ 。
D1118H-F：5’-TGTCTTTGTTTCAAATGGCACACAC-3’ ；
D1118H-R：5’-GGTTGCAAAGGATCATAAACTGTGT-3’ 。
by utilizing the detection primer, the invention provides a novel coronavirus point mutation S gene identification kit based on a high-resolution melting curve, which comprises the following components:
1) the primers specifically identify the S genes aiming at four mutation sites of N501Y, A570D, S982A and D1118H.
N501Y-F：5’-AGCACACCTTGTAATGGTGTTGAAG-3’ ；
N501Y-R：5’-TCCACAAACAGTTGCTGGTGCATGT-3’ 。
A570D-F：5’-GTTTAACAGGCACAGGTGTTCTTAC-3’ ；
A570D-R：5’-CACCACCAAAAGAACATGGTGTAAT-3’ 。
S982A-F：5’-CAAGCTTTAAACACGCTTGTTAAAC-3’ ；
S982A-R：5’-TGTCTGCAAACTTTGAAGTCTGCCT-3’ 。
D1118H-F：5’-TGTCTTTGTTTCAAATGGCACACAC-3’ ；
D1118H-R：5’-GGTTGCAAAGGATCATAAACTGTGT-3’ 。
2) PCR mixed solution: FastStart Taq DNA polymerase, reaction buffer solution, dNTP mixed solution and ResoLight saturated fluorescent dye;
3) a novel coronavirus S gene pUC57 vector recombinant plasmid containing N501Y, A570D, S982A and D1118H mutation sites;
4）ddH2O；
5）25 mM MgCl2。
the invention also provides a novel coronavirus point mutation S gene identification method based on a high-resolution melting curve, which comprises the following steps of:
1) high resolution melting curve template: preparation of recombinant plasmid of novel coronavirus S gene pUC57 vector containing four mutation sites of N501Y, A570D, S982A and D1118H:
the sequence of the novel coronavirus S gene (GeneID: 43740568) was obtained from NCBI website and the mutated gene was modified according to N501Y, A570D, S982A, D1118H mutation sites. The S gene sequence containing the mutation site is subjected to gene synthesis by the company of Biotechnology engineering (Shanghai) Ltd and constructed into a pUC57 plasmid to obtain a recombinant plasmid of the mutant S gene pUC57 vector.
2) High resolution melting curve reaction system:
the 30. mu.L reaction system included: 0.6. mu.L of primer F (10 μm working concentration), 0.6. mu.L of primer R (10 μm working concentration), 2. mu.L of template, 3. mu.L of reaction buffer, 0.5. mu.L of FastStart Taq DNA polymerase, 2. mu.L of dNTP mixture, MgCl2 3.6 μL，ResoLight saturated fluorescent dye 1 uL, ddH2O 16.7 μL。
3) High resolution melting curve reaction program:
pre-denaturation at 94 ℃ for 3 min. Denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles. Extension at 72 ℃ for 3 min.
The high resolution melting curve was run directly after the PCR was finished. Denaturation at 95 ℃ for 1 min. Cooling at 40 deg.C for 1 min. The temperature is continuously increased to 65-95 ℃ (1 ℃ is increased each time, and 25 fluorescence signals are collected). Cooling at 40 deg.C for 10 s. The high resolution melting curves were analyzed using LightCycler 480 Gene Scanning Software.
The invention is a 4 kinds of point mutation S gene discrimination kits of new coronavirus based on high resolution melting curve, its characteristic is that designing the specificity amplification primer according to 4 different mutation sites of new coronavirus S gene, can distinguish N501Y, A570D, S982A, D1118H mutation sites of new coronavirus S gene area, compare with existing identification means, the advantage of the invention lies in:
1) the novel coronavirus S gene region N501Y, A570D, S982A and D1118H mutations can be rapidly distinguished by PCR without gene sequencing.
2) The primer designed by the invention has high sensitivity and strong specificity.
3) The operation is simple, the speed is high, and whether the S gene region of the sample generates the four mutations can be determined after the experiment is completed.
4) The PCR product does not need post-treatment, realizes real closed-tube operation, thereby reducing the pollution risk, and is very suitable for the rapid detection of a large number of samples.
Drawings
FIG. 1: PCR result chart of four novel coronavirus mutant S genes.
FIG. 2: four novel coronavirus mutant S gene multicomponent maps.
FIG. 3: high resolution melting profiles of the four novel coronavirus mutant S genes relative to a negative control. (the abscissa is temperature, and the ordinate is relative fluorescence intensity), wherein the four fluctuation curves are four mutant genes respectively, and the straight line is a negative control.
Detailed Description
The invention is further described below by way of examples.
The invention provides a novel coronavirus point mutation S gene identification kit based on a high-resolution melting curve, which comprises the following components:
1) the S gene specific identification primers of N501Y, A570D, S982A and D1118H site mutation.
N501Y-F：5’-AGCACACCTTGTAATGGTGTTGAAG-3’ ；
N501Y-R：5’-TCCACAAACAGTTGCTGGTGCATGT-3’ 。
A570D-F：5’-GTTTAACAGGCACAGGTGTTCTTAC-3’ ；
A570D-R：5’-CACCACCAAAAGAACATGGTGTAAT-3’ 。
S982A-F：5’-CAAGCTTTAAACACGCTTGTTAAAC-3’ ；
S982A-R：5’-TGTCTGCAAACTTTGAAGTCTGCCT-3’ 。
D1118H-F：5’-TGTCTTTGTTTCAAATGGCACACAC-3’ ；
D1118H-R：5’-GGTTGCAAAGGATCATAAACTGTGT-3’ 。
2) PCR mixed solution: FastStart Taq DNA polymerase, reaction buffer, dNTP mixed solution and ResoLight saturated fluorescent dye
3) A novel coronavirus S gene pUC57 vector recombinant plasmid containing N501Y, A570D, S982A and D1118H mutation sites;
4）ddH2O；
5）25 mM MgCl2。
the invention also provides a novel coronavirus point mutation S gene identification method based on a high-resolution melting curve, which comprises the following steps of:
1) high resolution melting curve template: preparation of recombinant plasmid of novel coronavirus S gene pUC57 vector containing N501Y, A570D, S982A and D1118H mutation sites:
the sequence of the novel coronavirus S gene (GeneID: 43740568) was obtained from NCBI website and the mutated gene was modified according to N501Y, A570D, S982A, D1118H mutation sites. The S gene sequence containing the mutation site is subjected to gene synthesis by the company of Biotechnology engineering (Shanghai) Ltd and constructed into a pUC57 plasmid to obtain a recombinant plasmid of the mutant S gene pUC57 vector.
2) High resolution melting curve reaction system:
the 30. mu.L reaction system included: 0.6. mu.L of primer F (10 μm working concentration), 0.6. mu.L of primer R (10 μm working concentration), 2. mu.L of template, 3. mu.L of reaction buffer, 0.5. mu.L of FastStart Taq DNA polymerase, 2. mu.L of dNTP mixture, MgCl2 3.6 μ L of ResoLight saturated fluorescent dye 1 μ L, ddH2O 16.7 μL。
3) High resolution melting curve reaction program:
pre-denaturation at 94 ℃ for 3 min. Denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles. Extension at 72 ℃ for 3 min.
The high resolution melting curve was run directly after the PCR was finished. Denaturation at 95 ℃ for 1 min. Cooling at 40 deg.C for 1 min. The temperature is continuously increased to 65-95 ℃ (1 ℃ is increased each time, and 25 fluorescence signals are collected). Cooling at 40 deg.C for 10 s. The high resolution melting curves were analyzed using LightCycler 480 Gene Scanning Software.
4) As a result: as shown in FIG. 1, four novel coronavirus mutant S genes all amplified bands, and the sizes of the bands accord with the target bands. And (4) sequencing the genes and comparing by using NCBI Blast, wherein the comparison result shows that the gene mutation at the corresponding site is correct. The LightCycler 480 Gene Scanning Software was used to analyze the high resolution melting curve, and as shown in FIGS. 2 and 3, the curves of the four novel coronavirus mutant S genes were found to have significant differences: the dissolution temperature of the D1118H mutant S gene was 79.1 ℃, the dissolution temperature of the N501Y mutant S gene was 79.6 ℃, the dissolution temperature of the S982A mutant S gene was 80.8 ℃, and the dissolution temperature of the A570D mutant S gene was 81.6 ℃. Therefore, the novel coronavirus S gene with 4 different mutations can be effectively distinguished through the difference of the dissolving temperature.
5) And (4) conclusion: the invention provides a novel method for rapidly detecting the novel coronavirus mutant S gene by using a high-resolution melting curve, which is used for identifying four novel coronavirus mutant S genes for the first time. The kit is simple to operate and high in speed, PCR products do not need post-treatment, different mutations can be distinguished through different dissolving temperatures, gene sequencing is not needed, and closed-tube operation is really realized.
It is to be understood that the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and those skilled in the art should understand that they can make various changes, modifications, additions and substitutions within the spirit and scope of the present invention.
Claims (3)
1. The S gene specific identification primers of the novel coronavirus N501Y, A570D, S982A and D1118H site mutation based on a high-resolution melting curve have the following sequences:
N501Y-F：5’-AGCACACCTTGTAATGGTGTTGAAG-3’ ；
N501Y-R：5’-TCCACAAACAGTTGCTGGTGCATGT-3’ ；
A570D-F：5’-GTTTAACAGGCACAGGTGTTCTTAC-3’ ；
A570D-R：5’-CACCACCAAAAGAACATGGTGTAAT-3’ ；
S982A-F：5’-CAAGCTTTAAACACGCTTGTTAAAC-3’ ；
S982A-R：5’-TGTCTGCAAACTTTGAAGTCTGCCT-3’ ；
D1118H-F：5’-TGTCTTTGTTTCAAATGGCACACAC-3’ ；
D1118H-R：5’-GGTTGCAAAGGATCATAAACTGTGT-3’ 。
2. the novel coronavirus point mutation S gene identification kit based on the high-resolution melting curve comprises the following components:
1) s gene specific identification primers of N501Y, A570D, S982A and D1118H site mutation;
N501Y-F：5’-AGCACACCTTGTAATGGTGTTGAAG-3’ ；
N501Y-R：5’-TCCACAAACAGTTGCTGGTGCATGT-3’ ；
A570D-F：5’-GTTTAACAGGCACAGGTGTTCTTAC-3’ ；
A570D-R：5’-CACCACCAAAAGAACATGGTGTAAT-3’ ；
S982A-F：5’-CAAGCTTTAAACACGCTTGTTAAAC-3’ ；
S982A-R：5’-TGTCTGCAAACTTTGAAGTCTGCCT-3’ ；
D1118H-F：5’-TGTCTTTGTTTCAAATGGCACACAC-3’ ；
D1118H-R：5’-GGTTGCAAAGGATCATAAACTGTGT-3’ ；
2) PCR mixed solution: FastStart Taq DNA polymerase, reaction buffer solution, dNTP mixed solution and ResoLight saturated fluorescent dye;
3) a novel coronavirus S gene pUC57 vector recombinant plasmid containing N501Y, A570D, S982A and D1118H mutation sites;
4）ddH2O；
5）25 mM MgCl2。
3. a novel coronavirus point mutation S gene identification method based on a high-resolution melting curve comprises the following steps:
1) high resolution melting curve template: a novel coronavirus S gene pUC57 vector recombinant plasmid containing N501Y, A570D, S982A and D1118H mutation sites; preparation of recombinant plasmid:
obtaining a novel coronavirus S gene (GeneID: 43740568) sequence from an NCBI website, and modifying a mutant gene according to mutation sites of N501Y, A570D, S982A and D1118H; carrying out gene synthesis on an S gene sequence containing a mutation site and constructing the S gene sequence to a pUC57 plasmid to obtain a mutation S gene pUC57 vector recombinant plasmid;
2) high resolution melting curve reaction system:
the 30. mu.L reaction system included: 0.6. mu.L of primer F (10 μm working concentration), 0.6. mu.L of primer R (10 μm working concentration), 2. mu.L of template, 3. mu.L of reaction buffer, 0.5. mu.L of FastStart Taq DNA polymerase, 2. mu.L of dNTP mixture, MgCl2 3.6 μ L of ResoLight saturated fluorescent dye 1 μ L, ddH2O 16.7 μL；
3) High resolution melting curve reaction program:
pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extending for 3min at 72 ℃;
directly running a high-resolution melting curve after PCR is finished; denaturation at 95 deg.C for 1 min; cooling at 40 deg.C for 1 min; continuously heating to 65-95 ℃ (heating to 1 ℃ every time, collecting 25 fluorescence signals); cooling at 40 deg.C for 10 s; the high resolution melting curves were analyzed using LightCycler 480 Gene Scanning Software.
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| CN113699276A (en) * | 2021-08-24 | 2021-11-26 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Novel coronavirus 5-point mutation S gene identification kit based on high-resolution melting curve and identification method thereof |
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| CN113699276B (en) * | 2021-08-24 | 2024-02-02 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Novel coronavirus 5 point mutation S gene identification kit based on high-resolution melting curve and identification method thereof |
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