CN114150088A - New coronavirus gene single base mutation detection method and application thereof - Google Patents
New coronavirus gene single base mutation detection method and application thereof Download PDFInfo
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Abstract
The invention discloses a new coronavirus gene single base mutation detection method and application thereof, wherein the new coronavirus gene single base mutation detection method is based on polyethylene glycol assistance and realizes the detection of SARS-CoV-2 single base mutation conditions through hairpin probes PS-HP1 and HP 2. The method has simple operation, short detection time, can directly detect SARS-CoV-2 single base mutant gene samples, does not need strict thermal cycle process in reaction, has good specificity, is not easy to be influenced by other nucleic acids, has high sensitivity, has the detection limit of 4fM, and has good linearity between 10nM and 10 fM.
Description
Technical Field
The invention belongs to the field of gene detection, and particularly relates to a single base mutation detection method of a novel coronavirus gene and application thereof.
Background
A novel coronavirus (SARS-CoV-2) is an RNA virus with extremely strong hazard and strong mutation capability. It is widely monitored in various countries and regions around the world that SARS-CoV-2S protein is easy to generate amino acid mutation, especially the amino acid mutation in the receptor binding region or monoclonal antibody binding site causes the change of the transmission and pathogenicity of virus and partial immune escape. Taking the new crown virus delta variant as an example, the infectivity and the transmission capability of the new crown virus delta variant are obviously enhanced, and the spread is rapid since the first discovery. Therefore, in order to reduce the propagation risk, the method makes early warning for virus variation, realizes the real-time mutation detection of SARS-CoV-2 gene, and has important significance for epidemic prevention and control, epidemiological investigation and the like.
At present, SARS-CoV-2 gene mutation detection mainly relies on gene sequencing technology. The technology analyzes SARS-CoV-2 full-length genome, obtains gene mutation information of the whole genome, and is used for disease treatment, epidemiological analysis, etc. Although the existing sequencing technology has high accuracy and sensitivity and can comprehensively reflect the genetic information of pathogens, the existing sequencing technology usually needs to rely on a precise and expensive instrument, the detection is long in time consumption, the sequencing result depends on analysis and interpretation of professionals, the quantity of detected samples is relatively small, the existing sequencing technology is difficult to apply to large-scale clinical detection and on-site quick detection, the purposes of large-scale popularization and quick diagnosis cannot be realized, and hysteresis exists for monitoring novel variation and mutation of epidemic viruses (such as SARS-CoV-2).
The real-time fluorescence quantitative PCR technology is one of the most recommended SARS-CoV-2 detection methods of the WHO at present, and is also the gold standard for SARS-CoV-2 detection at present. However, the real-time fluorescent quantitative PCR can not directly detect the SARS-CoV-2 mutation site, but only can be used for the qualitative diagnosis or the quantitative detection of SARS-CoV-2, and on the basis, the SARS-CoV-2 mutation site of a subject is detected by combining the gene sequencing technology. The loop-mediated isothermal amplification technology is also one of the technologies commonly used for detecting SARS-CoV-2, and has the problems that the gene mutation site cannot be directly determined and a sequencing technology is required to be assisted.
Therefore, in order to monitor the development trend of the virus more effectively and understand the variation condition of the virus, the development of a detection method which is simple and convenient to operate and sensitive in detection and can be used for detecting single base mutation in a gene has great significance for epidemiological monitoring and population health monitoring.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. The single base mutation detection method of the new coronavirus gene can quickly and efficiently detect the single base mutation of the SARS-CoV-2 gene, has sensitive detection and good specificity, and has very important promotion effect on screening and monitoring of a variant strain of the new coronavirus.
In the first aspect of the invention, a hairpin probe for detecting single-base mutation of the novel coronavirus is provided, and the hairpin probe comprises PS-HP1 and HP 2.
According to a first aspect of the invention, in some embodiments of the invention, the PS-HP1 contains, in order, a hairpin complementary sequence, a new coronavirus targeting sequence, and a new coronavirus single base mutation recognition site. The HP2 in turn contains a hairpin complement and a novel coronavirus targeting sequence.
Wherein the hairpin complementary sequence comprises a hairpin complementary sequence to form a stem-loop structure; the new coronavirus single base mutation recognition site is used for distinguishing wild type new coronavirus from single base mutation type new coronavirus.
In some embodiments of the invention, the nucleotide sequence of PS-HP1 is: PS-HP 1:
wherein, 1 to 20 th bases of 5' end of PS-HP1 are addedTAGAATTCTAATGAATTCTA) Carrying out the thiophosphorylation modification.
In the present example, the phosphorothioate modification service of PS-HP1 was provided by Biotechnology (Shanghai) Inc.
The nucleotide sequence of the HP2 is as follows: HP 2:
the 5' end of the amino acid is Phosphorylated (PO)4) And (5) modifying.
Wherein, the only thickened parts of the PS-HP1 and HP2 are new coronavirus targeting sequences targeting SARS-CoV-2 gene.
The underlined parts of PS-HP1 and HP2 are hairpin complements to form stem-loop structures.
The last base C at the 3' end of PS-HP1 is a new coronavirus single base mutation recognition site.
According to a first aspect of the invention, in some embodiments of the invention, the novel single base mutant strain of coronaviruses comprises SARS-CoV-2D 614G.
In some embodiments of the invention, the SARS-CoV-2D614G is preferably SARS-CoV-2D614G comprising a signature sequence as shown in SEQ ID NO. 3.
In a second aspect of the present invention, there is provided a novel reagent for detecting single base mutation of coronavirus, which comprises the hairpin probe according to the first aspect of the present invention.
According to a second aspect of the invention, in some embodiments of the invention, the new single base mutant strain of coronavirus comprises SARS-CoV-2D 614G.
In some embodiments of the invention, the SARS-CoV-2D614G is preferably SARS-CoV-2D614G comprising a signature sequence as shown in SEQ ID NO. 3.
According to a third aspect of the invention, a new coronavirus single base mutation detection kit is provided, wherein the detection kit contains the detection reagent according to the second aspect of the invention, polyethylene glycol and high-fidelity DNA ligase.
According to the third aspect of the present invention, in some embodiments of the present invention, the detection kit further comprises a high fidelity DNA ligase buffer and a signal amplification system buffer.
In some embodiments of the present invention, the signal amplification system buffer preferably comprises a DNA polymerase and a double-stranded DNA dye.
In some preferred embodiments of the present invention, the signal amplification system buffer is:
| components | Content (wt.) |
| 7-8U/. mu.L DNA polymerase | 0.45～0.5μL； |
| 25～30mM dNTPs | 0.45～0.5μL； |
| 10×SYBR Green |
2～2.5μL； |
| 500-550 mu M betaine | 0.8～1μL； |
| 10 × |
2～2.5μL； |
| Water (W) | Make up to 10 mu L |
In some more preferred embodiments of the present invention, the signal amplification system buffer is shown in table 2 of the specification.
According to a third aspect of the invention, in some embodiments of the invention, the new single base mutant strain of coronavirus comprises SARS-CoV-2D 614G.
In some embodiments of the invention, the SARS-CoV-2D614G is preferably SARS-CoV-2D614G comprising a signature sequence as shown in SEQ ID NO. 3.
In a fourth aspect of the present invention, there is provided a method for detecting single base mutation of a novel coronavirus, comprising the steps of:
mixing the hairpin probe, polyethylene glycol, high-fidelity DNA ligase and a high-fidelity DNA ligase buffer solution, incubating for 4-6 minutes at 94-96 ℃, then incubating for 19-21 minutes at 63-67 ℃, adding a signal amplification system buffer solution, incubating for 55-65 minutes at 63-67 ℃, detecting fluorescence intensity, and judging whether a detected sample contains a new coronavirus single-base mutant strain.
In some preferred embodiments of the present invention, the method for detecting single base mutation of the novel coronavirus comprises the following steps:
mixing the hairpin probe, polyethylene glycol, high-fidelity DNA ligase and a high-fidelity DNA ligase buffer solution, incubating for 5 minutes at 95 ℃, then incubating for 20 minutes at 65 ℃, adding a signal amplification system buffer solution, incubating for 60 minutes at 65 ℃, detecting fluorescence intensity, and judging whether a detected sample contains the new coronavirus single-base mutant strain.
The inventor finds that the self-primer thiophosphoryl hairpin-mediated isothermal amplification without polyethylene glycol assistance can also be used for gene single-base mutation detection, but the detection effect on a low-concentration sample is not good, the minimum detection limit is about 5pM, the condition of low-concentration nucleic acid sample omission easily occurs, the self-primer thiophosphoryl hairpin-mediated isothermal amplification with polyethylene glycol assistance is obviously improved in detection sensitivity, the minimum detection limit is 4fM, and the evaluation detection precision is improved by about 100 times.
The detection principle of the single base mutation detection method of the novel coronavirus gene in the embodiment of the invention is as follows:
when SARS-CoV-2 with single base mutation is detected in the sample, the recognition sequences of the primer hairpin PS-HP1 and the SARS-CoV-2 target nucleic acid in HP2 are partially complementary with the SARS-CoV-2 target nucleic acid in the sample to form a dumbbell-shaped hairpin with a fracture (SARS-CoV-2 target nucleic acid-PS-HP 1-HP2 complex). Then, under the action of PEG 6000 and high fidelity DNA ligase (Hifi ligase) in the system, the SARS-CoV-2 target nucleic acid-PS-HP 1-HP2 complex catalyzes the 3 'hydroxyl of PS-HP1 and the 5' phosphate group of HP2 to form a phosphodiester bond, and connects the 3 'end of PS-HP1 and the 5' end of HP2 to form a complete dumbbell-shaped hairpin. After forming a complete dumbbell-shaped hairpin (PS-HP1-HP2 complex), the 3' end of the hairpin can be used as a self-primer, and the sequence of the self-PS-HP 1-HP2 complex is used as a template to extend along the 5' end under the action of DNA polymerase (Bst 2.0warmsmart DNA polymerase), so as to generate an extended hairpin structure (extension hairpin, EP1) containing a SARS-CoV-2 target nucleic acid double-stranded sequence, and the 5' end of the hairpin structure also contains 20 phosphorothioate-modified base sequences (from PS-HP1), which results in reduced stability, so that the 3' end of EP1 can be easily folded back to form the hairpin structure to form a new self-primer, and the extended self-sequence is used as a template and is extended by DNA polymerase, so as to generate an extended hairpin (EP2) with a longer stem again, which contains two SARS-CoV-2 target nucleic acid double-stranded sequences, and the 5' end of the extended product EP2 also contains 20 phosphorothioate-modified base sequences, the 3' end of the primer can be folded back to form a self-primer, and the primer is amplified again by using a longer self-sequence as a template, and the amplification is repeated in a circulating way to form an extension hairpin (EPx, x represents the number of repeated sections) with a plurality of sections of SARS-CoV-2 target nucleic acid double-stranded sequences. And the fluorescence intensity is obviously enhanced after the SYBR Green I is combined with the double chains of the amplification products, thereby determining the SARS-CoV-2 with single base mutation in the detection sample.
When SARS-CoV-2 does not exist or wild SARS-CoV-2 gene (no single base mutation) exists in the sample, the 3' end base of PS-HP1 is not paired with it, so that the subsequent reaction of forming phosphodiester bond between 3' end hydroxyl of PS-HP1 and 5' end phosphate group of HP2 will not proceed, and a complete dumbbell-shaped hairpin (PS-HP1-HP2 complex) can not be formed. The wild SARS-CoV-2 gene is locked and can not be used for subsequent nucleic acid amplification, SYBR Green I is in a free state, and the fluorescence intensity is extremely weak.
Therefore, the presence or absence of SARS-CoV-2 having a single-base mutation in a test sample can be determined by comparing the fluorescence intensities of the test sample and the negative control using the wild-type SARS-CoV-2 gene as the negative control.
According to a fourth aspect of the present invention, in some embodiments of the present invention, the criterion for determining whether the new single-base mutant strain of coronavirus is:
taking wild type new coronavirus as negative control, and if the fluorescence intensity of the detected sample is not obviously different from that of the wild type coronavirus, detecting that the detected sample does not contain the new coronavirus single-base mutant strain;
if the fluorescence intensity of the detected sample is obviously different from the fluorescence intensity of the wild type, the detected sample contains the new coronavirus single base mutant strain.
According to a fourth aspect of the invention, in some embodiments of the invention, the amplification system in the detection method is:
| 90～100nM PS-HP1 | 0.8～1.2μL； |
| 90～100nM HP2 | 0.8～1.2μL； |
| 10 XHi-Fi DNA ligase buffer solution | 0.8～1.2μL； |
| 30-40% of polyethylene glycol | 3.75～4μL； |
| High fidelity DNA ligase | 0.2～0.25μL； |
| 7-8U/. mu.L DNA polymerase | 0.45～0.5μL； |
| 25～30mM dNTPs | 0.45～0.5μL； |
| 10×SYBR Green |
2～2.5μL； |
| 500-550 mu M betaine | 0.8～1μL； |
| 10 × |
2～2.5μL； |
| Water (W) | Make up to 20 mu L |
。
In some preferred embodiments of the present invention, the amplification system in the detection method is:
| 100nM PS-HP1 | 1μL | |
| | 1μL | |
| 10×Hifi | 1μL | |
| 40% polyethylene glycol 6000 (final concentration in system is 15%) | 3.75μL | |
| Hifi ligase | 0.2μL | |
| Test sample | 1μL | |
| 8U/. mu.L Bst 2.0warmsmart DNA polymerase | 0.5μL | |
| 25mM dNTPs | 0.5μL | |
| 10×SYBR Green I | 2μL | |
| 500 μ M betaine | 1μL | |
| 10×isothemal amplification buffer | 2μL | |
| DEPC water | Make up to 20 mu L |
According to a fourth aspect of the invention, in some embodiments of the invention, the novel single base mutant strain of coronavirus comprises SARS-CoV-2D 614G.
In some embodiments of the invention, the SARS-CoV-2D614G is preferably SARS-CoV-2D614G comprising a signature sequence as shown in SEQ ID NO. 3.
In a fifth aspect of the invention, there is provided the use of the hairpin probe of the first aspect of the invention or the kit of the third aspect of the invention in the detection of single base mutations in a novel coronavirus.
According to a fifth aspect of the invention, in some embodiments of the invention, the new single base mutant strain of coronavirus comprises SARS-CoV-2D 614G.
In some embodiments of the invention, the SARS-CoV-2D614G is preferably SARS-CoV-2D614G comprising a signature sequence as shown in SEQ ID NO. 3.
The invention has the beneficial effects that:
1. the single base mutation detection method of the new coronavirus gene has the advantages of simple operation, short detection time, capability of directly detecting SARS-CoV-2 single base mutation gene samples, no need of strict thermal cycle process for reaction, good specificity, no easy influence of other nucleic acids, high sensitivity, detection limit of 4fM and good linearity between 10nM and 10 fM.
2. The reagent and the kit for detecting the single-base mutation of the new coronavirus gene can realize high-sensitivity detection based on a thiophosphorylation hairpin probe and a phosphorylation-modified hairpin probe, realize different and multi-scene applications by combining a real-time fluorescence quantitative PCR (polymerase chain reaction) instrument, a fluorescence spectrophotometer, a fluorescence enzyme labeling instrument and the like, and have extremely high practicability and universality.
Drawings
FIG. 1 is a schematic diagram of a method for detecting a single-base mutation in a novel coronavirus gene according to an embodiment of the present invention;
FIG. 2 is a real-time fluorescence intensity curve for the detection of SARS-CoV-2D614G target gene at different concentrations;
FIG. 3 is a standard curve of the method for detecting single base mutation of the novel coronavirus gene according to the embodiment of the present invention;
FIG. 4 is a real-time fluorescence intensity curve of different gene mutation samples;
FIG. 5 is a comparison of fluorescence intensities of different gene mutation samples;
FIG. 6 is a real-time fluorescence intensity curve of the detection method of single base mutation of the new coronavirus gene without using polyethylene glycol for the detection of SARS-CoV-2D614G target gene at different concentrations.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
New coronavirus gene single base mutation detection method
The single base mutation detection method of the novel coronavirus gene in the embodiment is to realize the detection of the single base mutation of the SARS-CoV-2 gene based on polyethylene glycol-assisted self-primer thiophosphorylation hairpin-mediated isothermal amplification.
The specific detection steps are as follows:
(1) designing a hairpin probe:
the hairpin probe in this example comprises PS-HP1 and HP2, wherein the nucleotide sequences are:
PS-HP1：
the 5' end of the amino acid is Phosphorylated (PO)4) Modifying;
HP2：
among them, only the bold portions of PS-HP1 and HP2 both targeted the SARS-CoV-2 gene. The bold and underlined portions of PS-HP1 and HP2 are hairpin complements to form stem-loop structures. The last base C at the 3' end of PS-HP1 is a single base recognition site.
Wherein, 1 to 20 th bases of 5' end of PS-HP1 are addedTAGAATTCTAATGAATTCTA) Carrying out the thiophosphorylation modification.
In the present example, the phosphorothioate modification service of PS-HP1 was provided by Biotechnology (Shanghai) Inc.
In this example, the SARS-CoV-2 gene is specifically SARS-CoV-2D 614G.
The target sequence of SARS-CoV-2D614G is: 5' -CAGGTTGCTGTTCTTTATCAGGGTGTTAACTGCACAGAA-3(SEQ ID NO. 3). The target sequence is located at 23381-23419 site bases of S protein of SARS-CoV-2D 614G.
(2) And (3) detection of a sample to be detected:
referring to the system shown in Table 1, the components except the buffer solution of the signal amplification system were mixed, incubated at 95 ℃ for 5 minutes, then at 65 ℃ for 20 minutes, the buffer solution of the signal amplification system was added, and the reaction was carried out at 65 ℃ for 60 minutes to detect the fluorescence intensity.
TABLE 1
Among them, 10 Xhifi ligand buffer and Hifi ligand are both from New Eangland Biolabs (New Eangland, USA).
The signal amplification system buffer was obtained by mixing the components shown in Table 2.
TABLE 2
| Components | Content (wt.) |
| 8U/. mu.L Bst 2.0warmsmart DNA polymerase | 0.5μL |
| 25mM dNTPs | 0.5μL |
| 10×SYBR Green I | 2μL |
| 500 μ M betaine | 1μL |
| 10×isothemal amplification buffer | 2μL |
| DEPC water | 4μL |
| Total of | 10μL |
Of these, 10 × Isothemal amplification buffer is available from New eagland Biolabs (New eagland, USA)
The detection principle of the single base mutation detection method of the novel coronavirus gene in the embodiment is as follows:
the detection schematic diagram of the single base mutation detection method of the novel coronavirus gene in this example is shown in FIG. 1.
When SARS-CoV-2 with single base mutation is detected in the sample, the recognition sequences of the primer hairpin PS-HP1 and the SARS-CoV-2 target nucleic acid in HP2 are partially complementary with the SARS-CoV-2 target nucleic acid in the sample to form a dumbbell-shaped hairpin with a fracture (SARS-CoV-2 target nucleic acid-PS-HP 1-HP2 complex). Then, the SARS-CoV-2 target nucleic acid-PS-HP 1-HP2 complex catalyzes the 3 'hydroxyl of PS-HP1 to form a phosphodiester bond with the 5' phosphate group of HP2 under the action of PEG 6000 and high fidelity DNA ligase (Hifi ligase) in the system, and connects the 3 'end of PS-HP1 and the 5' end of HP2 to form a complete dumbbell-shaped hairpin (HP1-HP2 complex). After forming a complete dumbbell-shaped hairpin, the stem-loop structure of the self-primer of HP2 will be extended along the 5' end of the HP1-HP2 complex by the action of DNA polymerase (Bst 2.0warmsmart DNA polymerase) to form an extended hairpin structure (EP1) containing a SARS-CoV-2 target nucleic acid duplex sequence, because the 5' end of EP1 contains 20 phosphorothioate-modified base sequences, which results in reduced stability, so that the 3' end of EP1 can easily fold back to form a hairpin structure, thereby forming a new self-primer, and the extended self-sequence (EP1) is used as a template to extend along the 5' end to generate an extended hairpin (EP2) containing two SARS-CoV-2 target nucleic acid duplex sequences, because the 5' end of the obtained EP2 still has 20 phosphorothioate-modified base sequences, therefore, after the extension of EP2 is completed, the 3' end is folded back again to form a hairpin structure, so as to form a new self-primer, and the cycle is repeated, thereby generating a long and multi-segment extended hairpin (EPx) amplification product containing a SARS-CoV-2 target nucleic acid double-stranded sequence long stem. And the fluorescence intensity of the SYBR Green I is obviously enhanced after the SYBR Green I is combined with the amplification product, so that the SARS-CoV-2 with single base mutation in the detection sample is determined.
When SARS-CoV-2 does not exist or wild SARS-CoV-2 gene (no single base mutation) exists in the sample, the 3' end base of PS-HP1 is not paired with it, so that the subsequent reaction of forming phosphodiester bond between the 3' end hydroxyl of PS-HP1 and the 5' end phosphate group of HP2 will not proceed, and a complete dumbbell-shaped hairpin (HP1-HP2 complex) can not be formed. The wild SARS-CoV-2 gene is locked and can not be used for subsequent nucleic acid amplification, SYBR Green I is in a free state, and the fluorescence intensity is extremely weak.
Therefore, the presence or absence of SARS-CoV-2 having a single-base mutation in a test sample can be determined by comparing the fluorescence intensities of the test sample and the negative control using the wild-type SARS-CoV-2 gene as the negative control.
Test for verifying detection effect
(1) Sensitivity:
the assay system was constructed according to the method in the above example, wherein the assay samples were SARS-CoV-2D614G at seven gradient concentrations of 0, 10fM, 1pM, 10pM, 100pM, 1nM and 10nM, respectively. The negative control was water.
The results of the tests are shown in FIGS. 2 and 3.
As can be seen from FIG. 2, the mutant target gene SARS-CoV-2D614G with different concentrations has a good fluorescence intensity gradient corresponding to it, indicating that the method in the above example has good detection linearity. Further, the fluorescence intensity of the SARS-CoV-2D614G with seven gradient concentrations of 0, 10fM, 1pM, 10pM, 100pM, 1nM and 10nM at 60 minutes is counted, and the linear relation among the seven concentrations detected is good, and the correlation coefficient is more than 0.98, which shows that the single base mutation detection method of SARS-CoV-2 gene based on the polyethylene glycol assisted autophosphorylation hairpin-mediated isothermal amplification of autophosphorylation hairpin of autophosphorylation primers in the above example is good in linearity in the range of 10fM-10 nM. The minimum detection limit of 4fM can be calculated by using the fluorescence intensity of the negative control at 60 minutes, which is the minimum detection limit, as a negative control and using 3 times the fluorescence intensity of the negative control as a y value by a linear range formula. Compared with the detection method in the prior art, the detection method for the single base mutation of the SARS-CoV-2 gene based on the polyethylene glycol assisted autophosphorylation hairpin-mediated isothermal amplification in the embodiment has higher detection precision and the detection sensitivity is improved by about 100 times.
(2) Specificity:
the test system was constructed according to the method in the above example, wherein the test samples (each at a concentration of 10nM) were as follows:
SARS-CoV-2D614G：5’-CAGGTTGCTGTTCTTTATCAGGGTGTTAACTGCACAGAA-3’；
wild type SARS-CoV-2 (WH): 5' -CAGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAA-3’(SEQ ID NO.4)；
Among them, underlined parts of the mutant gene (D614G) and the wild-type gene (WH) are opposite single-base mutation sites.
Mismatch sequence 1 (M1): 5' -CAGGTTGCTGTTCTTTATCAGAATGTTAACTGCACAGAA-3’(SEQ ID NO.5)；
Mismatch sequence 2 (M2): 5' -CAGGTTGCTGTTCTTTATCCGAATGTTAACTGCACAGAA-3’(SEQ ID NO.6)；
Mismatch sequence 3 (M3): 5' -CAGGTTGCTGTTCTTTATCCGGATCTGAACTGCACAGAA-3’(SEQ ID NO.7)。
Among these, underlined portions in the mismatch sequences 1 to 3 are mismatch bases corresponding to the mismatch sequences 1 to 3 and the mutant gene (D614G).
The negative control is water instead of the target nucleic acid, and the other components are unchanged.
The results of the tests are shown in FIGS. 4 and 5.
As shown in FIGS. 4 and 5, the fluorescence intensity of the wild-type gene WH was about 15% of that of the perfectly matched D614G with 10nM of the mutant target gene SARS-CoV-2D614G as X-axis and fluorescence intensity as Y-axis. This shows that the single base mutation detection method based on the polyethylene glycol assisted autophosphorylation hairpin mediated isothermal amplification SARS-CoV-2 gene in the above examples can well distinguish SARS-CoV-2 wild type gene and SARS-CoV-2D614G single base mutant gene, and can well shield the interference of interference sequence. The results also suggest that the method is expected to be applied to direct detection of all mutation sites of SARS-CoV-2 related Variant (VOC) and concerned Variant (VOI).
Comparative example 1
The detection method is the same as the single base mutation detection method of the new coronavirus gene in the above example, except that in comparative example 1, 40% of polyethylene glycol 6000 was replaced with an equal amount of water.
In comparative example 1, the test samples were SARS-CoV-2D614G at different concentrations (0, 10pM, 100pM, 1nM and 10 nM).
The negative control was water.
The results of the detection are shown in FIG. 6.
It can be found that when the SARS-CoV-2D614G with different concentrations is detected by using the SARS-CoV-2 gene single base mutation detection method of autophosphorylation hairpin-mediated isothermal amplification without adding polyethylene glycol, the fluorescence intensity corresponding to the low-concentration SARS-CoV-2D614G mutant gene can not form a gradient, which indicates that the sensitivity of autophosphorylation hairpin-mediated isothermal amplification can be obviously reduced and the low-concentration SARS-CoV-2D614G single base mutant gene can not be effectively distinguished without the assistance of polyethylene glycol.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Guangdong university of medical science
<120> detection method for single base mutation of new coronavirus gene and application thereof
<130>
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 37
<212> DNA
<213> Artificial sequence
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tagaattcta atgaattcta ttctgtgcag ttaacac 37
<210> 2
<211> 42
<212> DNA
<213> Artificial sequence
<400> 2
cctgataaag aacagcaacc tggttagtgg aataccacta ac 42
<210> 3
<211> 39
<212> DNA
<213> SARS-CoV-2 D614G
<400> 3
caggttgctg ttctttatca gggtgttaac tgcacagaa 39
<210> 4
<211> 39
<212> DNA
<213> SARS-CoV-2
<400> 4
caggttgctg ttctttatca ggatgttaac tgcacagaa 39
<210> 5
<211> 39
<212> DNA
<213> Artificial sequence
<400> 5
caggttgctg ttctttatca gaatgttaac tgcacagaa 39
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<211> 39
<212> DNA
<213> Artificial sequence
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caggttgctg ttctttatcc gaatgttaac tgcacagaa 39
<210> 7
<211> 39
<212> DNA
<213> Artificial sequence
<400> 7
caggttgctg ttctttatcc ggatctgaac tgcacagaa 39
Claims (10)
1. A hairpin probe for detecting single-base mutation of new coronavirus is characterized in that the hairpin probe comprises PS-HP1 and HP 2;
the PS-HP1 sequentially contains a hairpin complementary sequence, a new coronavirus targeting sequence and a new coronavirus single base mutation recognition site;
the 5' hairpin complementary sequence of PS-HP1 is modified by phosphorothioate;
the HP2 sequentially contains a hairpin complementary sequence and a new coronavirus targeting sequence;
the hairpin complementary sequence contains a hairpin complementary sequence so as to form a stem-loop structure;
the new coronavirus single base mutation recognition site is used for distinguishing wild type new coronavirus from single base mutation type new coronavirus.
2. A hairpin probe according to claim 1,
the nucleotide sequence of the PS-HP1 is as follows: 5'-TAGAATTCTAATGAATTCTATTCTGTGCAGTTAACAC-3' (SEQ ID NO. 1);
the nucleotide sequence of the HP2 is as follows: 5'-CCTGATAAAGAACAGCAACCTGGTTAGTGGAATACCACTAAC-3' (SEQ ID NO. 2).
3. A reagent for detecting single base mutation of a novel coronavirus, which comprises the hairpin probe of any one of claims 1 to 2.
4. A kit for detecting single base mutation of a novel coronavirus, which is characterized by comprising the detection reagent as claimed in claim 3, polyethylene glycol and high-fidelity DNA ligase.
5. The detection kit according to claim 4, further comprising a signal amplification system buffer and a high-fidelity DNA ligase buffer;
wherein, the buffer solution of the signal amplification system preferably comprises DNA polymerase and double-stranded DNA dye.
6. A method for detecting single base mutation of new coronavirus comprises the following steps:
mixing the hairpin probe as claimed in any one of claims 1 to 2, polyethylene glycol, high-fidelity DNA ligase and a high-fidelity DNA ligase buffer solution, incubating at 94-96 ℃ for 4-6 minutes, then incubating at 63-67 ℃ for 19-21 minutes, adding a signal amplification system buffer solution, incubating at 63-67 ℃ for 55-65 minutes, detecting fluorescence intensity, and judging whether a detected sample contains a new corona virus single-base mutant strain.
7. The method of claim 6, wherein the determination criterion for the presence or absence of a single-base mutant strain of a novel coronavirus is as follows:
taking wild type new coronavirus as negative control, and if the fluorescence intensity of the detected sample is not obviously different from that of the wild type coronavirus, detecting that the detected sample does not contain the new coronavirus single-base mutant strain;
if the fluorescence intensity of the detected sample is obviously different from the fluorescence intensity of the wild type, the detected sample contains the new coronavirus single base mutant strain.
8. The detection method according to claim 6, wherein the amplification system in the detection method is:
9. the detection method as claimed in claim 6, wherein the new coronavirus single base mutant strain comprises SARS-CoV-2D614G, and the SARS-CoV-2D614G is preferably SARS-CoV-2D614G containing the characteristic sequence shown in SEQ ID NO. 3.
10. Use of the hairpin probe of any one of claims 1 to 2 or the kit of any one of claims 4 to 5 for the detection of single base mutations in a novel coronavirus.
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