CN113699276B - Novel coronavirus 5 point mutation S gene identification kit based on high-resolution melting curve and identification method thereof - Google Patents
Novel coronavirus 5 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 5 point mutation S gene identification kit based on a high-resolution melting curve and an identification method thereof, belonging to the field of biological medicine. The kit is simple to operate, high in reaction speed, free of post-treatment of PCR products, and capable of being used for identifying novel coronavirus S genes with 5 different site mutations, and has operability and reproducibility.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a novel coronavirus point mutation S gene identification kit based on a high-resolution melting curve, a detection method and application thereof.
Background
The epidemic situation of the new coronaries and pneumonia brings huge disasters to human society since the outbreak, and the epidemic situation of the new coronaries and pneumonia is not effectively controlled until now. The rapid transmission of the novel coronavirus not only seriously affects the development of the global public health industry, but also puts forward higher requirements on port health quarantine and epidemic prevention and control. Gene mutations are a common phenomenon in the natural evolution of viruses, and studies have shown that novel coronavirus mutants may make them more transmissible or produce the ability to evade certain antibody immune responses. The spike protein (S) on the surface of the novel coronavirus plays a very important role in virus transmission, and the rapid identification of the S gene mutant virus is not only beneficial to epidemic prevention and control, but also beneficial to further monitoring the transmission and evolution progress of the virus. Therefore, the development of a safe, rapid, accurate and sensitive rapid detection method for mutant genes is urgently needed for port new crown quarantine so as to realize rapid identification of novel coronavirus S gene mutants.
In recent years, with the development of molecular biology technology, single-strand conformational polymorphism analysis (SSCP), high performance liquid chromatography, and molecular approaches such as first, second, third and metagenomic sequencing have been successfully applied to the identification of known or unknown mutant genes. However, these identification methods have disadvantages: SSCP has higher sensitivity, but can only distinguish normal chains and mutant chains through electrophoresis, the mutation type and mutation gene locus still need to be further sequenced to be determined, and the electrophoresis condition is strict; the high performance liquid chromatography can only check whether mutation exists or not, the mutation type can not be detected, and the result judgment is easy to be in error; 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 shortcomings of the above methods, in order to safely, rapidly, accurately and sensitively realize the molecular identification of mutant genes, a new molecular identification means needs to be searched.
Disclosure of Invention
Aiming at the above defects or improvement demands 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 of mutation S genes, is used for carrying out mutation gene identification based on different forms of melting curves formed by different single nucleotide melting temperatures, has the advantages of simple operation, high speed, low cost, high flux, accurate result, no limitation of detection sites, no need of post-treatment of PCR products, capability of detecting single base differences and the like, realizes real closed-tube operation, reduces 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 S gene identification primers for the mutation of the position of the N501Y, A570D, P681H, S982A, D1118H of the coronavirus 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’ 。
P681H-F：5’-AACAACTCATATGAGTGTGACATAC-3’ ；
P681H-R：5’-AGCAACTGAATTTTCTGCACCAAGT-3’ 。
S982A-F：5’-CAAGCTTTAAACACGCTTGTTAAAC-3’ ；
S982A-R：5’-TGTCTGCAAACTTTGAAGTCTGCCT-3’ 。
D1118H-F：5’-TGTCTTTGTTTCAAATGGCACACAC-3’ ；
D1118H-R：5’-GGTTGCAAAGGATCATAAACTGTGT-3’ 。
the invention provides a novel coronavirus point mutation S gene identification kit based on a high-resolution melting curve by using the detection primer, which comprises the following components:
1) S gene specific identification primers for five mutation sites of N501Y, A570D, P681H, S982A, D1118H.
N501Y-F：5’-AGCACACCTTGTAATGGTGTTGAAG-3’ ；
N501Y-R：5’-TCCACAAACAGTTGCTGGTGCATGT-3’ 。
A570D-F：5’-GTTTAACAGGCACAGGTGTTCTTAC-3’ ；
A570D-R：5’-CACCACCAAAAGAACATGGTGTAAT-3’ 。
P681H-F：5’-AACAACTCATATGAGTGTGACATAC-3’ ；
P681H-R：5’-AGCAACTGAATTTTCTGCACCAAGT-3’ 。
S982A-F：5’-CAAGCTTTAAACACGCTTGTTAAAC-3’ ；
S982A-R：5’-TGTCTGCAAACTTTGAAGTCTGCCT-3’ 。
D1118H-F：5’-TGTCTTTGTTTCAAATGGCACACAC-3’ ；
D1118H-R：5’-GGTTGCAAAGGATCATAAACTGTGT-3’ 。
2) PCR mixture: fastStart Taq DNA polymerase, reaction buffer, dNTP mixed solution, resoLight saturated fluorescent dye;
3) A novel coronavirus S gene pUC57 vector recombinant plasmid containing N501Y, A570D, P681H, S982A, D1118H mutation site;
4）ddH 2 O；
5）25 mM MgCl 2 。
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:
1) High resolution melting curve template: preparation of novel coronavirus S gene pUC57 vector recombinant plasmid containing five mutation sites of N501Y, A570D, P681H, S982A, D1118H:
the novel coronavirus S gene (GeneID: 43740568) sequence was obtained from NCBI website and the mutant gene was modified according to the mutation site of N501Y, A570D, P681H, S982A, D1118H. The S gene sequence containing the mutation site is submitted to the engineering (Shanghai) Co.Ltd for gene synthesis and constructed to pUC57 plasmid, and the mutant S gene pUC57 vector recombinant plasmid is obtained.
2) High resolution melting curve reaction system:
the 30. Mu.L reaction system comprises: primer F0.6. Mu.L (10 μm working concentration), primer R0.6. Mu.L (10 μm working concentration), template 2. Mu.L, reaction buffer 3. Mu.L, fastStart Taq DNA polymerase 0.5. Mu.L, dNTP mix 2. Mu.L, mgCl 2 3.6 Mu L, resoLight saturated fluorochrome 1 mu L, ddH 2 O 16.7 μL。
3) High resolution melting curve reaction procedure:
pre-denatured at 94 ℃ for 3 min. Denaturation at 94℃for 30s, annealing at 52℃for 30s, extension at 72℃for 1min for 30 cycles. Extending at 72℃for 3 min.
After the PCR is finished, the high-resolution melting curve is directly operated. Denaturation at 95℃for 1min. Cooling at 40deg.C for 1min. The temperature is continuously raised to 65-95 ℃ and 25 fluorescence signals are collected at 1 ℃ each time. Cooled at 40℃for 10s. The high resolution melting curve was analyzed using the LightCycler 480 Gene Scanning Software software.
The invention is a new coronavirus 5 point mutation S gene identification kit based on high resolution melting curve, characterized in that designing specific amplification primer according to 5 different mutation sites of new coronavirus S gene can identify mutation site of new coronavirus S gene region N501Y, A570D, P681H, S982A, D1118H, compared with existing identification means, the invention has the advantages that:
1) The novel coronavirus S gene region N501Y, A570D, P681H, S982A, D1118H mutation can be rapidly distinguished by PCR without the need for gene sequencing.
2) The primer designed by the invention has high sensitivity and strong specificity.
3) The operation is simple and the speed is high, and the experiment can be completed to determine whether the S gene region of the sample generates the five mutations.
4) The PCR product does not need post-treatment, realizes real closed-tube operation, reduces pollution risk, and is very suitable for rapid detection of a large number of samples.
Drawings
Fig. 1: five novel coronavirus mutant S gene PCR results.
Fig. 2: five novel coronavirus mutant S gene multicomponent maps.
Fig. 3: high resolution melting profiles of five novel coronavirus mutant S genes relative to negative controls. (temperature on the abscissa and relative fluorescence intensity on the ordinate), wherein five fluctuation curves are five mutant genes, respectively, and a 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) S gene specific identification primer of N501Y, A570D, P681H, S982A, D1118H site mutation.
N501Y-F：5’-AGCACACCTTGTAATGGTGTTGAAG-3’ ；
N501Y-R：5’-TCCACAAACAGTTGCTGGTGCATGT-3’ 。
A570D-F：5’-GTTTAACAGGCACAGGTGTTCTTAC-3’ ；
A570D-R：5’-CACCACCAAAAGAACATGGTGTAAT-3’ 。
P681H-F：5’-AACAACTCATATGAGTGTGACATAC-3’ ；
P681H-R：5’-AGCAACTGAATTTTCTGCACCAAGT-3’ 。
S982A-F：5’-CAAGCTTTAAACACGCTTGTTAAAC-3’ ；
S982A-R：5’-TGTCTGCAAACTTTGAAGTCTGCCT-3’ 。
D1118H-F：5’-TGTCTTTGTTTCAAATGGCACACAC-3’ ；
D1118H-R：5’-GGTTGCAAAGGATCATAAACTGTGT-3’ 。
2) PCR mixture: fastStart Taq DNA polymerase, reaction buffer solution, dNTP mixed solution, resoLight saturated fluorescent dye
3) A novel coronavirus S gene pUC57 vector recombinant plasmid containing N501Y, A570D, P681H, S982A, D1118H mutation site;
4）ddH 2 O；
5）25 mM MgCl 2 。
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:
1) High resolution melting curve template: preparation of novel coronavirus S gene pUC57 vector recombinant plasmid containing N501Y, A570D, P681H, S982A, D1118H mutation site:
the novel coronavirus S gene (GeneID: 43740568) sequence was obtained from NCBI website and the mutant gene was modified according to the mutation site of N501Y, A570D, P681H, S982A, D1118H. The S gene sequence containing the mutation site is submitted to the engineering (Shanghai) Co.Ltd for gene synthesis and constructed to pUC57 plasmid, and the mutant S gene pUC57 vector recombinant plasmid is obtained.
2) High resolution melting curve reaction system:
the 30. Mu.L reaction system comprises: primer F0.6. Mu.L (10 μm working concentration), primer R0.6. Mu.L (10 μm working concentration), template 2. Mu.L, reaction buffer 3. Mu.L, fastStart Taq DNA polymerase 0.5. Mu.L, dNTP mix 2. Mu.L, mgCl 2 3.6 Mu L, resoLight saturated fluorochrome 1 mu L, ddH 2 O 16.7 μL。
3) High resolution melting curve reaction procedure:
pre-denatured at 94 ℃ for 3 min. Denaturation at 94℃for 30s, annealing at 52℃for 30s, extension at 72℃for 1min for 30 cycles. Extending at 72℃for 3 min.
After the PCR is finished, the high-resolution melting curve is directly operated. Denaturation at 95℃for 1min. Cooling at 40deg.C for 1min. The temperature is continuously raised to 65-95 ℃ and 25 fluorescence signals are collected at 1 ℃ each time. Cooled at 40℃for 10s. The high resolution melting curve was analyzed using the LightCycler 480 Gene Scanning Software software.
4) Results: as shown in FIG. 1, five novel coronavirus mutant S genes all amplify bands, and the sizes of the bands meet the target bands. The gene is sequenced and compared by NCBI Blast, and the comparison result shows that the gene mutation at the corresponding site is correct. The high resolution melting curve is analyzed by using the LightCycler 480 Gene Scanning Software software, as shown in figures 2 and 3, and the curve difference of five novel coronavirus mutant S genes is found to be obvious: the dissolution temperature of the D1118H mutant S gene was 79.1℃and the dissolution temperature of the N501Y mutant S gene was 79.6℃and 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℃and the dissolution temperature of the P681H mutant S gene was 82.5 ℃. Thus, the novel coronavirus S gene with 5 different mutations can be effectively distinguished by the difference of the dissolution temperature.
5) Conclusion: the invention firstly utilizes a high-resolution melting curve to identify five novel coronavirus mutant S genes, and provides a novel method for rapidly detecting the novel coronavirus mutant S genes through the high-resolution melting curve. The kit is simple to operate, high in speed, and capable of distinguishing different mutations through different dissolution temperatures without gene sequencing, and the PCR product does not need post-treatment, so that closed tube operation is truly realized.
It should be understood that the above description is not intended to limit the invention to the particular embodiments disclosed, but to limit the invention to the particular embodiments disclosed, and that various changes, modifications, additions and substitutions can be made by those skilled in the art without departing from the spirit and scope of the invention.
Sequence listing
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<120> novel coronavirus 5-point mutation S gene identification kit based on high resolution melting curve and identification method thereof
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Claims (2)
1. The S gene specific identification multiplex primer of the novel coronavirus N501Y, A570D, P681H, S982A, D1118H site mutation based on the high resolution melting curve has the following sequence:
N501Y-F：5’-AGCACACCTTGTAATGGTGTTGAAG-3’ ；
N501Y-R: 5'-TCCACAAACAGTTGCTGGTGCATGT-3'; and
A570D-F：5’-GTTTAACAGGCACAGGTGTTCTTAC-3’ ；
a570D-R: 5'-CACCACCAAAAGAACATGGTGTAAT-3'; and
P681H-F：5’-AACAACTCATATGAGTGTGACATAC-3’ ；
P681H-R: 5'-AGCAACTGAATTTTCTGCACCAAGT-3'; and
S982A-F：5’-CAAGCTTTAAACACGCTTGTTAAAC-3’ ；
S982A-R: 5'-TGTCTGCAAACTTTGAAGTCTGCCT-3'; and
D1118H-F：5’-TGTCTTTGTTTCAAATGGCACACAC-3’ ；
D1118H-R：5’-GGTTGCAAAGGATCATAAACTGTGT-3’。
2. novel coronavirus point mutation S gene identification kit based on high-resolution melting curve comprises the following components:
1) Multiplex primer is identified specifically by S gene of N501Y, A570D, P681H, S982A, D1118H site mutation;
N501Y-F：5’-AGCACACCTTGTAATGGTGTTGAAG-3’ ；
N501Y-R: 5'-TCCACAAACAGTTGCTGGTGCATGT-3'; and
A570D-F：5’-GTTTAACAGGCACAGGTGTTCTTAC-3’ ；
a570D-R: 5'-CACCACCAAAAGAACATGGTGTAAT-3'; and
P681H-F：5’-AACAACTCATATGAGTGTGACATAC-3’ ；
P681H-R: 5'-AGCAACTGAATTTTCTGCACCAAGT-3'; and
S982A-F：5’-CAAGCTTTAAACACGCTTGTTAAAC-3’ ；
S982A-R: 5'-TGTCTGCAAACTTTGAAGTCTGCCT-3'; and
D1118H-F：5’-TGTCTTTGTTTCAAATGGCACACAC-3’ ；
D1118H-R：5’-GGTTGCAAAGGATCATAAACTGTGT-3’ ；
2) PCR mixture: fastStart Taq DNA polymerase, reaction buffer, dNTP mixed solution, resoLight saturated fluorescent dye;
3) A novel coronavirus S gene pUC57 vector recombinant plasmid containing N501Y, A570D, P681H, S982A, D1118H mutation site;
4）ddH 2 O；
5）25 mM MgCl 2 。
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|---|---|---|---|---|
| CN112813203A (en) * | 2021-03-02 | 2021-05-18 | 常州国药医学检验实验室有限公司 | New coronavirus nucleic acid and mutant gene detection kit based on fluorescent quantitative PCR technology and application thereof |
| CN113005226A (en) * | 2021-02-07 | 2021-06-22 | 利多(香港)有限公司 | Oligonucleotide and kit for detecting SARS-CoV-2 |
| CN113025760A (en) * | 2021-05-11 | 2021-06-25 | 常州国药医学检验实验室有限公司 | Multiple fluorescence quantitative PCR technology-based novel coronavirus mutant sequence detection technology and application thereof |
| CN113046475A (en) * | 2021-01-14 | 2021-06-29 | 复旦大学附属华山医院 | Primer composition and kit for rapidly detecting mutant type novel coronavirus |
| CN113736915A (en) * | 2021-08-05 | 2021-12-03 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Novel coronavirus 3-point mutation S gene identification kit based on high-resolution melting curve and identification method thereof |
| CN114085924A (en) * | 2021-08-05 | 2022-02-25 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Novel coronavirus 4-point mutation S gene identification kit based on high-resolution melting curve and identification method thereof |
-
2021
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113046475A (en) * | 2021-01-14 | 2021-06-29 | 复旦大学附属华山医院 | Primer composition and kit for rapidly detecting mutant type novel coronavirus |
| CN113005226A (en) * | 2021-02-07 | 2021-06-22 | 利多(香港)有限公司 | Oligonucleotide and kit for detecting SARS-CoV-2 |
| CN112813203A (en) * | 2021-03-02 | 2021-05-18 | 常州国药医学检验实验室有限公司 | New coronavirus nucleic acid and mutant gene detection kit based on fluorescent quantitative PCR technology and application thereof |
| CN113025760A (en) * | 2021-05-11 | 2021-06-25 | 常州国药医学检验实验室有限公司 | Multiple fluorescence quantitative PCR technology-based novel coronavirus mutant sequence detection technology and application thereof |
| CN113736915A (en) * | 2021-08-05 | 2021-12-03 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Novel coronavirus 3-point mutation S gene identification kit based on high-resolution melting curve and identification method thereof |
| CN114085924A (en) * | 2021-08-05 | 2022-02-25 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Novel coronavirus 4-point mutation S gene identification kit based on high-resolution melting curve and identification method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Recurrent emergence and transmission of a SARS-CoV-2 spike deletion H69/V70;Kemp等;bioRxiv;参见全文 * |
| The N501Y spike substitution enhances SARS-CoV-2 transmission;Liu等;bioRxiv;参见全文 * |
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