CN113249522A - Method for detecting SARS-CoV-2variant strain nucleic acid and its application - Google Patents
Method for detecting SARS-CoV-2variant strain nucleic acid and its application Download PDFInfo
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Abstract
The invention discloses a method for detecting SARS-CoV-2variant strain nucleic acid and its application. The inventor finds that the quenching capacity of guanine is obviously reduced after nucleotide G and nucleotide C are complementarily combined, and designs a self-quenching probe capable of identifying single base difference by utilizing the finding, thereby breaking through the technical bottleneck that the fluorescence PCR technology is difficult to realize single base identification. The probe for detecting a nucleotide mutation in a nucleic acid molecule provided by the present invention is designed based on the nucleotide mutation, wherein a nucleotide G corresponding to the nucleotide mutation is designated as a target G; the probe is labeled with a fluorescent dye, and the fluorescent dye is labeled on adjacent nucleotides of the target G. Furthermore, the invention can realize the single base rapid identification of the new coronavirus variant strain, and has great value for the rapid identification of the novel coronavirus pneumonia variant strain and the epidemic situation prevention and control.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a method for detecting SARS-CoV-2variant strain nucleic acid and application thereof.
Background
The novel coronavirus (SARS-CoV-2) is a new respiratory tract pathogen capable of causing the novel human coronavirus pneumonia (COVID-19), belongs to the beta-coronavirus with the severe acute respiratory syndrome coronavirus (SARS-CoV) and the middle east respiratory syndrome coronavirus (MERS-CoV), and has higher infectivity and certain lethality rate.
The spinous process protein (S) is the most prominent glycoprotein on the surface of SARS-CoV-2 and is composed of two subunits, S1 and S2. The Receptor Binding Domain (RBD) at the end of S1 initiates viral replication by binding to the cell surface receptor angiotensin converting enzyme 2(ACE2), which infects cells. Blocking RBD binding to the receptor inhibits viral replication, and therefore the S and RBD proteins are the target antigens for most vaccines. Moreover, most of the potent monoclonal antibodies found also recognize mainly the RBD region. Coronaviruses belong to the RNA viruses, and therefore have high variability. There have been more and more variant strains discovered, including b.1.1.7(501y.v1) first found in the uk, b.1.351(501y.v2) found in south africa, p.1(501y.v3) found in brazil, b.1.617(452r.v3) found in india, etc. These variant strains are more infectious than the initially isolated wild-type strains, and mutations occurring in the RBD region affect the protective effect of the vaccine, causing viral immune escape. Research has shown that although mutations at multiple sites, including K417N/T, E484K and N501Y, are found in RBD region, the mutation at E484 site can seriously affect the protective effect of vaccine and Antibody, compared with other site mutations, resulting in more than 10 times reduction of vaccine neutralization protection, and multiple approved monoclonal Antibody drugs also lose the neutralizing activity to the variant strain at the site (Antibody Resistance of SARS-CoV-2Variants B.1.351and B.1.1.7.Nature. (2021)). Therefore, the rapid identification of the E484 site variation has important significance for vaccination, epidemic prevention and control and epidemiological investigation. It was demonstrated that there are mutant strains with immune escape, including B.1.351, P.1 and B.1.617, all containing E484 site variation, and that the escape responses of these strains were directly related to E484 site (SARS-CoV-2spike E484K mutation recovery. Lancet Microbe (2021)).
At present, the identification of variant strains is mainly performed by a sequencing technology, but the sequencing technology is difficult to realize high-throughput rapid determination and has high requirements on technology and equipment, so that the large-scale popularization and application of the variant strains are limited. The fluorescence PCR technology is the most widely applied nucleic acid detection technology, and the nucleic acid detection of SARS-CoV-2 is mainly based on TaqMan fluorescence PCR technology at present. At present, county-level detection organizations in China all have conditions for developing fluorescence PCR, and if the fluorescence PCR can be used for realizing the rapid identification of variant strains, the detection is the optimal selection. However, the TaqMan fluorescent PCR technology cannot realize the identification of single base, and some researchers try to perform sequence analysis on a variant strain, and find that the variant strain has 3675-3677 base deletion on ORF1a gene in about 80% of B.1.351 besides the most concerned E484 site mutation, so that a TaqMan fluorescent PCR detection method (PCR assay to enhance viral surface mutation for SARS-CoV-2variants of company. medium Rxiv. (2021)) is designed based on the base deletion. Since the B.1.1.7 variant strain also has the base deletion, and about 0.03 percent of wild strains also have the deletion, the application of the detection method cannot effectively distinguish E484 site mutant strains. Moreover, the B.1.1.7 variant strain does not influence the immune protection effect of the vaccine, so the method has great application limitation.
It is believed in the prior art that guanine quenches the Fluorescence of its adjacent fluorescent dye by means of electron transfer (Fluorescence-quenching phenomenon by photoinduced electron transfer between fluorescent dye and nuclear base. anal Sci. (2001).17,155-160,10.2116/analsci.17.155.) thus TaqMan probes require that the fluorophore not be labeled on nucleotide G.
Disclosure of Invention
The invention aims to provide a method for detecting SARS-CoV-2variant strain nucleic acid and its application.
The invention provides a probe for detecting nucleotide mutation in nucleic acid molecules, which is a probe A or a probe B;
for the probe A, the nucleotide mutation is a mutation related to nucleotide G, namely the nucleotide G is used before mutation or the nucleotide G is used after mutation; the probe is a forward hybridization probe, is designed based on the nucleotide mutation, wherein a nucleotide G corresponding to the nucleotide mutation is designated as a target G; the probe is marked with a fluorescent dye, and the fluorescent dye is marked on adjacent nucleotides of the target G;
for the probe B, the nucleotide mutation is a mutation related to nucleotide C, namely the nucleotide C is obtained before mutation or the nucleotide C is obtained after mutation; the probe is a reverse hybridization probe, designed based on the nucleotide mutation, wherein a nucleotide G corresponding to the nucleotide mutation is designated as a target G; the probe is labeled with a fluorescent dye, and the fluorescent dye is labeled on adjacent nucleotides of the target G.
The probes are useful for nucleic acid detection, such as hybridization, fluorescent PCR, and the like.
Such fluorescent dyes include, but are not limited to, FAM, HEX, VIC, TAMRA, CY5, and the like.
The adjacent nucleotide may be the adjacent nucleotide of the 5 'end of the target G, and may also be the adjacent nucleotide of the 3' end of the target G.
Target G may be located at the 3 'end of the probe, at the 5' end of the probe, or within the probe.
The nucleic acid molecule may be a DNA molecule or an RNA molecule.
The invention also provides a primer probe group for detecting nucleotide mutation in nucleic acid molecules, which comprises the probe and a specific primer pair; the target sequence of the specific primer pair comprises a sequence corresponding to the probe.
By adopting the primer probe set, the amplification of the template nucleic acid and the identification of the single base can be realized by utilizing a PCR system.
When the probe in the primer probe set is a probe A, the nucleotide mutation is related to the mutation of the nucleotide G, namely the nucleotide G is obtained before the mutation or the nucleotide G is obtained after the mutation.
When the probe in the primer probe group is the probe B, the nucleotide mutation is the mutation related to the nucleotide C, namely the nucleotide C is obtained before the mutation or the nucleotide C is obtained after the mutation.
The nucleic acid molecule may be a DNA molecule or an RNA molecule.
The invention also provides a fluorescent PCR method (method A) for identifying nucleotide mutations in nucleic acid molecules, comprising the following steps: using a test DNA as a template, and carrying out PCR amplification by adopting the primer probe group; identifying nucleotide mutations in the nucleic acid molecule according to the amplification curve. The nucleic acid molecule may be a DNA molecule.
When the probe in the primer probe set is a probe A, the nucleotide mutation is related to the mutation of the nucleotide G, namely the nucleotide G is obtained before the mutation or the nucleotide G is obtained after the mutation.
When the probe in the primer probe group is the probe B, the nucleotide mutation is the mutation related to the nucleotide C, namely the nucleotide C is obtained before the mutation or the nucleotide C is obtained after the mutation.
The invention also protects a fluorescent PCR method (method B) for identifying nucleotide mutations in a nucleic acid molecule, comprising the steps of: carrying out PCR amplification in a system containing reverse transcriptase by using the primer probe group by using test RNA as a template; identifying nucleotide mutations in the nucleic acid molecule according to the amplification curve. The nucleic acid molecule may be an RNA molecule.
When the probe in the primer probe set is a probe A, the nucleotide mutation is related to the mutation of the nucleotide G, namely the nucleotide G is obtained before the mutation or the nucleotide G is obtained after the mutation.
When the probe in the primer probe group is the probe B, the nucleotide mutation is the mutation related to the nucleotide C, namely the nucleotide C is obtained before the mutation or the nucleotide C is obtained after the mutation.
In any of the above methods, the concentration of the forward primer in the reaction system for PCR amplification may be 0.5-10mM (e.g., 0.8. mu.M), and the concentration of the backward primer may be 0.5-10mM (e.g., 0.8. mu.M).
In any of the above methods, the concentration of the probe in the reaction system for PCR amplification may be 0.01 to 0.4. mu.M (e.g., 0.2. mu.M).
Since the probe employed in the present invention emits fluorescence without resorting to exonuclease treatment, it is different from the TaqMan PCR technique in which only a PCR enzyme possessing exonuclease activity can be selected. In any of the above methods, the DNA polymerase used for PCR amplification includes, but is not limited to, Taq polymerase, Pfu polymerase, KOD polymerase, Tth polymerase, Deep Vent polymerase, and Bst polymerase. Any DNA polymerase capable of effecting nucleic acid amplification can be used.
The invention also protects the application of the probe or the primer probe group in the preparation of a kit; the kit is used for identifying nucleotide mutations in nucleic acid molecules. The nucleic acid molecule may be a DNA molecule or an RNA molecule.
When the probe is a probe A, the nucleotide mutation is related to the mutation of the nucleotide G, namely the nucleotide G is obtained before the mutation or the nucleotide G is obtained after the mutation.
And when the probe is a probe B, the nucleotide mutation is the mutation related to the nucleotide C, namely the nucleotide C is obtained before mutation or the nucleotide C is obtained after mutation.
When the probe in the primer probe set is a probe A, the nucleotide mutation is related to the mutation of the nucleotide G, namely the nucleotide G is obtained before the mutation or the nucleotide G is obtained after the mutation.
When the probe in the primer probe group is the probe B, the nucleotide mutation is the mutation related to the nucleotide C, namely the nucleotide C is obtained before the mutation or the nucleotide C is obtained after the mutation.
Illustratively, any of the nucleic acid molecules described above can be a virus-specific nucleic acid molecule.
Illustratively, the virus may be a novel coronavirus.
Illustratively, the nucleotide mutation in any of the nucleic acid molecules described above can be an E484 site mutation.
Meaning of E484 site mutation: the nucleotide corresponding to the E484 site of the novel coronavirus wild-type strain is the nucleotide G, the E484 site of the E484 variant strain is mutated into K or Q, and the corresponding nucleotide is mutated into the nucleotide A or the nucleotide C.
Illustratively, the probe may be probe SARS2dP-1 or probe SARS2 dP-2.
Probe SARS2dP-1 is as follows (sequence 3 in the sequence table):
the 3' terminal nucleotide is the target G and the TAMRA label is on the adjacent T upstream of the target G.
Probe SARS2dP-2 is as follows (sequence 4 in the sequence table):
the 2 nd nucleotide from the 3' end is the target G, and the TAMRA label is located on the adjacent T downstream of the target G.
Illustratively, the specific primer pairs are as follows:
SARS2F (upstream primer, sequence 5 of sequence listing): 5'-GCTGCGTTATAGCTTGGAATTC-3', respectively;
SARS2B (downstream primer, sequence 6 of sequence listing): 5'-GGGTTGGAAACCATATGATTGT-3' are provided.
The invention also provides a kit for detecting E484 site variation of the novel coronavirus, which comprises a specific probe; the specific probe is designed based on the E484 site variation of the novel coronavirus, wherein the specific probe has a nucleotide G corresponding to the E484 site variation of the novel coronavirus and is named as a target G; the specific probe is labeled with a fluorescent dye, and the fluorescent dye is labeled on adjacent nucleotides of the target G.
Such fluorescent dyes include, but are not limited to, FAM, HEX, VIC, TAMRA, CY5, and the like.
The adjacent nucleotide may be the adjacent nucleotide of the 5 'end of the target G, and may also be the adjacent nucleotide of the 3' end of the target G.
Target G may be located at the 3 'end of the specific probe, at the 5' end of the specific probe, or inside the specific probe.
The kit also comprises a specific primer pair; the target sequence of the specific primer pair comprises a sequence corresponding to the specific probe.
The kit further comprises a universal probe; the universal probe is designed based on a conserved region of a novel coronavirus, which has a nucleotide G and is labeled with a fluorescent dye on a nucleotide adjacent to the nucleotide G.
Such fluorescent dyes include, but are not limited to, FAM, HEX, VIC, TAMRA, CY5, and the like.
The adjacent nucleotide may be the adjacent nucleotide at the 5 'end of the nucleotide G, and may also be the adjacent nucleotide at the 3' end of the nucleotide G.
The nucleotide G may be located at the 3 'end of the universal probe, at the 5' end of the universal probe, or internally within the universal probe.
The target sequence of the specific primer pair comprises a sequence corresponding to the universal probe.
The fluorescent dye on the specific probe is different from the fluorescent dye on the universal probe.
Illustratively, the specific probe may be probe SARS2dP-1 or probe SARS2 dP-2.
Probe SARS2dP-1 is as follows (sequence 3 in the sequence table):
the 3' terminal nucleotide is the target G and the TAMRA label is on the adjacent T upstream of the target G.
Probe SARS2dP-2 is as follows (sequence 4 in the sequence table):
the 2 nd nucleotide from the 3' end is the target G, and the TAMRA label is located on the adjacent T downstream of the target G.
Illustratively, the specific primer pairs are as follows:
SARS2F (upstream primer, sequence 5 of sequence listing): 5'-GCTGCGTTATAGCTTGGAATTC-3', respectively;
SARS2B (downstream primer, sequence 6 of sequence listing): 5'-GGGTTGGAAACCATATGATTGT-3' are provided.
Illustratively, the universal probe is the probe SARS2 dP-U.
Probe SARS2dP-U is as follows (sequence 7 in the sequence table):
the 3' terminal nucleotide is the target G, and the FAM tag is located on the adjacent T upstream of the target G.
The invention also protects the application of (a) or (b) or (c) or (d) in preparing a kit for detecting E484 site variation of the novel coronavirus;
(a) the specific probe;
(b) the specific probe and the universal probe;
(c) the specific probe and the specific primer pair;
(d) the specific probe, the universal probe and the specific primer pair.
The invention also protects the application of (a) or (b) or (c) or (d) in preparing a kit for detecting the novel coronavirus E484 variant strain;
(a) the specific probe;
(b) the specific probe and the universal probe;
(c) the specific probe and the specific primer pair;
(d) the specific probe, the universal probe and the specific primer pair.
The inventor of the invention finds that when the nucleotide G is complementarily combined with the nucleotide C, the quenching capacity of guanine is obviously reduced, and further the inventor designs a self-quenching probe (the fluorescence of guanine quenching fluorescent dye and the fluorescence of fluorescent dye after complementary combination of guanine and cytosine) capable of identifying single base difference by utilizing the finding, so that the rapid identification of single base mutation can be realized, and the technical bottleneck that the single base identification is difficult to realize by a fluorescent PCR technology is broken through.
Furthermore, the invention can realize the rapid identification of the single base of the new coronavirus variant strain. Therefore, the method has great value for rapid identification of the variant strain of the novel coronavirus pneumonia and epidemic prevention and control.
Drawings
FIG. 1 is a schematic diagram of the working principle of the specific probe.
FIG. 2 is a schematic diagram of the working principle of the specific probe and the general probe.
FIG. 3 is a graph showing the results of example 2.
FIG. 4 is a graph showing the results of example 3.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The pGEM-T vector is a commercial vector (Cat. No.: A3600, Promega, USA). The working concentration of the primer/probe is the concentration of the primer/probe in the PCR reaction system. Unless otherwise stated, the quantitative tests in the following examples were performed in triplicate, and the results were averaged.
Example 1 novel findings on the resolution of guanine-quenching fluorescence and preliminary development of the protocol
Previous studies found that guanine can quench the labeled fluorophore (Fluorescence-quenching electron transfer beta. fluorescent dye and a nucleotide base. anal Sci 17,155-160(2001)), and that TaqMan probes were designed based on this theory that the fluorophore cannot be labeled on nucleotide G or its adjacent nucleotides. The inventors have found that the ability of guanine on an oligonucleotide to quench fluorescence is significantly reduced when the oligonucleotide is double-stranded with a complementary sequence.
Using the above-mentioned important findings, the inventors have arrived at a technical solution (which is applicable to mutations involving a nucleotide G or C, i.e., a nucleotide G or C before mutation or a nucleotide G or C after mutation; the probe is designed based on the nucleotide mutation, wherein a nucleotide G corresponding to the nucleotide mutation is designated as a target G): labeling a fluorescent dye on adjacent nucleotides of a target G in a probe, wherein the fluorescent dye on the probe does not emit fluorescence due to the quenching function of guanine; when the probe is combined with the complementary sequence, the quenching capability of guanine can be eliminated after the nucleotide C on the complementary sequence is combined with the target G on the probe, so that the fluorescent dye on the probe emits fluorescence; if the complementary sequence lacks nucleotide C and cannot bind to the target G on the probe, the probe will not fluoresce. Through the technical scheme, the single base of the nucleic acid can be identified, the rapid nucleic acid detection similar to the TaqMan fluorescent PCR technology can be realized by matching the fluorescent PCR technology, and the single base mutation identification function which is not possessed by the TaqMan technology is also possessed.
Example 2 design and characterization of SARS-CoV-2 self-quenching Probe
Design of probe
Using the protocol of example 1, a self-quenching probe was designed that recognized the E484 site of the variant strain. The nucleotide corresponding to the wild-type strain E484 site is G (guanine nucleotide), which is defined as target G; the E484 site of the variant strains B.1.351, P.1 and the like is mutated into K, and the corresponding nucleotide is A (adenine ribonucleotide); the E484 site of the B.1.617 mutant strain was mutated to Q, and the corresponding nucleotide was C (cytosine ribonucleotide). The target G may be designed at the 3 'end of the probe, the target G may be designed at the 5' end of the probe, or the target G may be designed inside the probe, and the final detection effect is not affected. The probe is designed according to the wild type strain E484 site, and a fluorescent group (such as FAM or TAMRA) is marked on the adjacent nucleotide of the target G. Ensuring that the probe has no obvious secondary structure such as a hairpin loop.
This embodiment provides only one probe sequence and labeling method, but is not limited to this design. Probes were designed based on the RBD gene sequences of the Wuhan-Hu-1 strain (GenBank: MN908947.3), the B.1.351 variant strain (GISAID EPI _ ISL _1250476), the P.1 variant strain (GISAID EPI _ ISL _1073034) and the B.1.617(GISAID EPI _ ISL _1360382), and fluorescent dyes were labeled on the adjacent nucleotides at the 5 'end or 3' end of the target G, respectively. Probe SARS2dP-1 and probe SARS2dP-2 are both specific probes for E484 site.
Probe SARS2dP-1 is as follows (sequence 3 in the sequence table):
target G is underlined and TAMRA is marked on the adjacent T upstream of target G (T is marked with a box).
Probe SARS2dP-2 is as follows (sequence 4 in the sequence table):
target G is underlined and TAMRA is marked on the adjacent T downstream of target G (the T is marked with a box).
The PCR amplification primers matched with the probe are as follows:
SARS2F (upstream primer, sequence 5 of sequence listing): 5'-GCTGCGTTATAGCTTGGAATTC-3', respectively;
SARS2B (downstream primer, sequence 6 of sequence listing): 5'-GGGTTGGAAACCATATGATTGT-3' are provided.
Secondly, identifying wild strains and variant strains by fluorescence PCR
The template solutions were respectively plasmid P0Solutions or plasmids PVAnd (3) solution. Plasmid POThe recombinant plasmid is obtained by cloning RBD gene (shown as sequence 1 in a sequence table) of the Wuhan-Hu-1 strain to pGEM-T vector. Plasmid PVThe recombinant plasmid is obtained by cloning RBD gene (shown as sequence 2 in a sequence table) of the B.1.351 variant strain into pGEM-T vector. DNA content in the template solution was 106Copy/ml or 104Copy/ml.
PCR amplification was performed. Three amplification systems are respectively adopted for PCR amplification. Both the amplification system used SARS2F (upstream primer) and SARS2B (downstream primer). The amplification system adopts probe SARS2dP-1 or probe SARS2 dP-2.
Composition of Taq amplification System (25. mu.l): reaction buffer solution, dNTP, Taq polymerase, an upstream primer, a downstream primer, a probe, template solution and water. The working concentration of the upstream primer is 0.8 mu M, the working concentration of the downstream primer is 0.8 mu M, and the working concentration of the probe is 0.2 mu M. In the reaction system, the amount of the template solution added was 5. mu.l. The reaction buffer, dNTP, and Taq polymerase were all provided by the commercial reagent Taq PCR mix (Beijing Quanji Co., Cat. No.: AS 111-01).
Composition of Pfu amplification system (25. mu.l): reaction buffer solution, dNTP, Pfu polymerase, upstream primer, downstream primer, probe, template solution and water. The working concentration of the upstream primer is 0.8 mu M, the working concentration of the downstream primer is 0.8 mu M, and the working concentration of the probe is 0.2 mu M. In the reaction system, the amount of the template solution added was 5. mu.l. The reaction buffer, dNTP, and Pfu polymerase were all provided by the commercial reagent Pfu PCR mix (Kyoto Kagaku Co., Ltd.; cat. AS 211-01).
Composition of KOD amplification system (25 μ l): reaction buffer solution, dNTP, KOD polymerase, upstream primer, downstream primer, probe, template solution and water. The working concentration of the upstream primer is 0.8 mu M, the working concentration of the downstream primer is 0.8 mu M, and the working concentration of the probe is 0.2 mu M. In the reaction system, the amount of the template solution added was 5. mu.l. The reaction buffer, dNTP, KOD polymerase were supplied from a commercial reagent KOD mix (Kyoto Kagaku Co., Ltd.; cat. AS 301-01).
The PCR amplification procedures used for all three amplification systems were as follows: 3min at 95 ℃; 95 ℃ 15s, 58 ℃ 30s (fluorescence collected), 40 cycles.
When the RBD gene of the Wuhan-Hu-1 strain is used as the target, no matter which amplification system is adopted, no matter which probe SARS2dP-1 or probe SARS2dP-2 is adopted, no matter which amplification system is adopted, the DNA content of the template solution is 106Copy/ml is also 104Copy/ml, all show sigmoidal amplification curves.
B.1.351 mutant strain RBD gene as target, regardless of whether probe SARS2dP-1 or probe SARS2dP-2 is adopted, regardless of which amplification system is adopted, regardless of whether the DNA content of template solution is 106Copy/ml is also 104Copy/ml, no amplification curve.
The results are shown in FIG. 3. In FIG. 3, the wild strain corresponds to plasmid POThe E484 variant strain corresponds to the plasmid PV。
Thirdly, judging results and analyzing principles
When the template contains RBD gene of new coronavirus, the specific primer can start PCR amplification. After the probe binds to the probe complementary sequence in the amplification sequence, the quenching ability of guanine is reduced, and fluorescence is emitted. The RBD gene of the Wuhan-Hu-1 strain can be completely and complementarily combined with the probe to emit fluorescence, so that an S-shaped amplification curve is formed. The RBD gene of the variant strain containing the E484 site mutation cannot be completely complementarily combined with the probe, so that the quenching capability of guanine cannot be influenced, fluorescence cannot be emitted, and an S-shaped amplification curve cannot be formed. Whether the amplified sequence contains the E484 site mutation can be judged by observing whether an amplification S-shaped curve is formed. The schematic diagram is shown in figure 1.
Example 3 establishment and verification of SARS-CoV-2 Dual Probe detection and mutant Strain identification System
Through the example 2, whether the RBD gene of the new coronavirus contains E484 site mutation or not can be quickly identified, but the E484 site mutation strain cannot be distinguished from a negative result only by the method. In the embodiment, a general probe is added, and a fluorescent group different from the specific probe is marked to establish a double-probe quenching fluorescent PCR technology, so that the nucleic acid detection of the new coronavirus can be realized, and the variant strain can be identified.
Design of probe
The probe designed in this example is consistent with the probe design strategy of example 2, and is also a guanine quenching probe. The probe is selected in the amplification sequence of the two primers, is not overlapped with the target sequence of the probe in the embodiment 2, is a new coronavirus conserved region, is completely complementary with the Wuhan-Hu-1 strain and the E484 site-containing variant strain, and is different from the probe in the embodiment 1 in the labeled fluorescent dye. The probe SARS2dP-U is a novel coronavirus universal probe.
Probe SARS2dP-U is as follows (sequence 7 in the sequence table):
target G is underlined and the FAM label is on the adjacent T upstream of target G (T is marked with a box).
Secondly, identifying wild strains and variant strains by fluorescence PCR
The template solution was the same as in example 2.
PCR amplification is carried out by adopting a Taq amplification system.
Composition of Taq amplification System (25. mu.l): reaction buffer solution, dNTP, Taq polymerase, SARS2F (upstream primer), SARS2B (downstream primer), probe SARS2dP-1, probe SARS2dP-U, template solution and water. The working concentration of SARS2F (upstream primer) is 0.8. mu.M, the working concentration of SARS2B (downstream primer) is 0.8. mu.M, the working concentration of probe SARS2dP-1 is 0.2. mu.M, and the working concentration of probe SARS2dP-U is 0.04. mu.M. In the reaction system, the amount of the template solution added was 5. mu.l. The reaction buffer, dNTP, and Taq polymerase were all provided by the commercial reagent Taq PCR mix (Beijing Quanji Co., Cat. No.: AS 111-01).
The PCR amplification procedure was the same as in example 2.
When the RBD gene of the Wuhan-Hu-1 strain is used as a target, the DNA content of a template solution is 106Copy/ml is also 104Copies/ml, either FAM or TAMRA fluorescence signals of the universal or specific probes, showed a sigmoidal amplification curve.
When the RBD gene of B.1.351 variant strain is taken as a target, no matter the DNA content of the template solution is 106Copy/ml is also 104Copy/ml, FAM fluorescence signals of the universal probes all show sigmoidal amplification curves.
When the RBD gene of B.1.351 variant strain is taken as a target, no matter the DNA content of the template solution is 106Copy/ml is also 104Copy/ml, no amplification curve was observed for TAMRA fluorescence signal of the specific probe.
The results are shown in FIG. 4. In FIG. 4, the wild strain corresponds to plasmid POThe E484 variant strain corresponds to the plasmid PV。
Thirdly, judging results and analyzing principles
When the template contains a new coronavirus RBD gene without E484 mutation, the specific probe and the general probe can emit fluorescence with different wavelengths, and two S-shaped amplification curves appear; when the template contains E484 mutant new coronavirus RBD gene, the general probe can emit fluorescence, the specific probe can not emit fluorescence, and 1S-shaped amplification curve appears; when the template does not contain a new coronavirus RBD gene, the two probes do not emit fluorescence, and an S-shaped amplification curve does not appear. Thereby realizing two functions of detecting the nucleic acid of the new coronavirus and identifying the variant strain at the same time. The schematic diagram is shown in fig. 2.
EXAMPLE 4 SARS-CoV-2 double Probe detection and mutant Strain identification System minimum detection Limit and specificity evaluation one, evaluation of minimum detection Limit
1. Preparation of Standard solutions
Taking the plasmid, and adopting T7 RiboMAXTMExpress Large Scale RNA Production System kit (Promega, cat # P1320) for in vitro transcription; then, DNase (Promega, cat # P1320) was added to the transcript to remove the remaining plasmid; then, the high-purity in vitro transcribed RNA was obtained by purification using RNeasy clean kit (Qiagen, cat # 74204).
The high-purity in vitro transcription RNA is quantified by a Nano-Drop 2000 nucleic acid quantifier (Thermo Fisher Scientific), RNA copy number concentration (copy number/ml) is calculated, and RNase-free ddH is used2And diluting with O to obtain standard solutions with different copy number concentrations.
The plasmids are respectively plasmid P0Or plasmid PV. Plasmid P0Performing the above steps to obtain RNA0Obtaining RNA correspondingly0And (4) standard solution. Plasmid PVPerforming the above steps to obtain RNAVObtaining RNA correspondinglyvAnd (4) standard solution.
2. Fluorescent PCR
The template solution is RNA0Standard solution or RNAvAnd (4) standard solution.
PCR amplification is carried out by adopting a Taq amplification system.
Composition of Taq amplification System (25. mu.l): reaction buffer solution, dNTP, Taq polymerase, reverse transcriptase, SARS2F (upstream primer), SARS2B (downstream primer), probe SARS2dP-1, probe SARS2dP-U, template solution and water. The working concentration of SARS2F (upstream primer) is 0.8. mu.M, the working concentration of SARS2B (downstream primer) is 0.8. mu.M, the working concentration of probe SARS2dP-1 is 0.2. mu.M, and the working concentration of probe SARS2dP-U is 0.04. mu.M. In the reaction system, the amount of the template solution added was 5. mu.l, and the amount of the reverse transcriptase added was 5U. The reaction buffer, dNTP, and Taq polymerase were all provided by the commercial reagent Taq PCR mix (Beijing Quanji Co., Cat. No.: AS 111-01). Reverse transcriptase: takara, cat No.: 639522.
water was used as a negative control instead of the standard solution;
the PCR amplification procedure was as follows: 15min at 50 ℃; 3min at 95 ℃; 10s at 95 ℃ and 30s at 58 ℃ (fluorescence collected), for 40 cycles.
Ct values the results are given in Table 1.
3. Comparative test
The template solution is RNA0Standard solution or RNAvAnd (4) standard solution.
PCR amplification is carried out by adopting a Taq amplification system.
Composition of Taq amplification System (25. mu.l): reaction buffer, dNTP, Taq polymerase, reverse transcriptase, SARS2F (upstream primer), SARS2B (downstream primer), TaqMan probe, template solution and water. The working concentration of SARS2F (upstream primer) was 0.8. mu.M, SARS2B (downstream primer) was 0.8. mu.M, and the working concentration of TaqMan probe was 0.2. mu.M. In the reaction system, the amount of the template solution added was 5. mu.l, and the amount of the reverse transcriptase added was 5U. The reaction buffer, dNTP, and Taq polymerase were all provided by the commercial reagent Taq PCR mix (Beijing Quanji Co., Cat. No.: AS 111-01). Reverse transcriptase: takara, cat No.: 639522.
TaqMan probe (SARS2TaqMan p): 5 '-FAM-ACCATTACAAGGTGTGCTACCGGCCTG-BHQ-3'.
Ct values the results are given in Table 1.
The universal probe and the specific probe have the lowest detection limit of 2 copies/response to the RBD gene of the Wuhan-Hu-1 strain. The universal probe has a minimum detection limit of 2 copies/response to the RBD gene of the B.1.351 variant strain. The lowest detection limit of the TaqMan probe for the RBD gene of the Wuhan-Hu-1 strain and the RBD gene of the B.1.351 variant strain is 2 copies/response.
TABLE 1
Second, evaluation of specificity
Different respiratory pathogens, including Influenza A Viruses H1N 1and H3N2 (described in Interaction amplification Viruses A/H1N1, A/H3N2, and B in Japan. International viral research and public health 16(2019)), Influenza B Viruses Yamagata and Victoria types (described in A Novel reduced specific nucleic acid PCR for distinguishing between Influenza B Viruses Yamagata and viral genes. journal of clinical virology (2020)), syncytial virus A/B types (described in BD: Novel multiplex-time RT-for synthesis virus of the same type 2 SARS virus, 2022N 1/2/3, SARS 3N 2022 and PCR for distinguishing between Influenza B Viruses), Influenza A/B types (described in SARS virus of the same type 2022, 20210, PCR for distinguishing between Influenza B Viruses), Influenza A/B types (described in PCR for distinguishing between Influenza B Viruses), Influenza B types B virus B and Influenza B virus B3, PCR for distinguishing between Influenza B Viruses B and Influenza B virus B (2. 3. M. A. 7. A. 7. 3. A. 7. infection of Influenza virus and Influenza virus (3. A. 7. origin. A. 7. infection, virus of infection, infection of Influenza virus, infection of Influenza virus, infection of Influenza virus, infection of Influenza virus, infection of Influenza virus, infection of infection, infection of infection, infection of virus, infection of virus, infection of virus, infection of infection, infection of infection, infection of virus, infection of the same type of virus, infection of virus, infection of virus, infection of the same type of virus, infection of virus, infection of virus, infection of the same, infection of virus, infection of the same type of virus, infection of, adenovirus type (described in Human adenovirus species in Children with access sensitivity in viruses. journal of Clinical visibility. the invention is also applicable to Clinical visibility 134,104716(2021)), Rhinovirus (Viral Loads and Disease visibility in Children with Rhinovirus-Associated viruses. visions 13(2021)), coronavirus NL63 and HKU1 (described in A patent with Human vironavir NL63 facial diagnosis with COF 19; Lesson attached for the diagnosis of infection of virus J2028. the invention is also applicable to the detection of specificity of adenovirus type (described in Human adenovirus in Clinical diagnosis of diagnosis J. 2028).
Extracting total RNA of the pathogen as a template solution. And (3) detecting according to the method 2 in the step one.
No amplification curve was observed for either the universal or specific probes.
The results prove that the detection method of the invention has no cross reaction with the pathogens.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
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Claims (10)
1. A probe for detecting nucleotide mutation in a nucleic acid molecule is a probe A or a probe B;
for the probe A, the nucleotide mutation is a mutation related to nucleotide G, namely the nucleotide G is used before mutation or the nucleotide G is used after mutation; the probe is a forward hybridization probe, is designed based on the nucleotide mutation, wherein a nucleotide G corresponding to the nucleotide mutation is designated as a target G; the probe is marked with a fluorescent dye, and the fluorescent dye is marked on adjacent nucleotides of the target G;
for the probe B, the nucleotide mutation is a mutation related to nucleotide C, namely the nucleotide C is obtained before mutation or the nucleotide C is obtained after mutation; the probe is a reverse hybridization probe, designed based on the nucleotide mutation, wherein a nucleotide G corresponding to the nucleotide mutation is designated as a target G; the probe is labeled with a fluorescent dye, and the fluorescent dye is labeled on adjacent nucleotides of the target G.
2. A primer probe set for detecting nucleotide mutations in a nucleic acid molecule comprising the probe of claim 1and a specific primer pair; the target sequence of the specific primer pair comprises a sequence corresponding to the probe.
3. A fluorescent PCR method for identifying nucleotide mutations in a nucleic acid molecule, comprising the steps of: performing PCR amplification by using the primer probe set of claim 2 using the test DNA as a template; identifying nucleotide mutations in the nucleic acid molecule according to the amplification curve.
4. A fluorescent PCR method for identifying nucleotide mutations in a nucleic acid molecule, comprising the steps of: performing PCR amplification in a reverse transcriptase-containing system using the primer probe set of claim 2 with the test RNA as a template; identifying nucleotide mutations in the nucleic acid molecule according to the amplification curve.
5. Use of the probe of claim 1 or the primer probe set of claim 2 in the preparation of a kit; the kit is used for identifying nucleotide mutations in nucleic acid molecules.
6. A kit for detecting E484 site variation of a novel coronavirus, which comprises a specific probe; the specific probe is designed based on the E484 site variation of the novel coronavirus, wherein the specific probe has a nucleotide G corresponding to the E484 site variation of the novel coronavirus and is named as a target G; the specific probe is labeled with a fluorescent dye, and the fluorescent dye is labeled on adjacent nucleotides of the target G.
7. The kit of claim 6, wherein: the kit also comprises a specific primer pair; the target sequence of the specific primer pair comprises a sequence corresponding to the specific probe.
8. The kit of claim 6 or 7, wherein: the kit further comprises a universal probe; the universal probe is designed based on a conserved region of a novel coronavirus, which has a nucleotide G and is labeled with a fluorescent dye on a nucleotide adjacent to the nucleotide G.
Use of (a) or (b) or (c) or (d) for the preparation of a kit for the detection of a variation in the E484 site of a novel coronavirus;
(a) a specific probe as described in claim 6;
(b) a specific probe as described in claim 6 and a universal probe as described in claim 8;
(c) a specific probe as described in claim 6 and a specific primer pair as described in claim 7;
(d) the specific probe as claimed in claim 6 and the universal probe as claimed in claim 8 and the specific primer pair as claimed in claim 7.
Use of (a) or (b) or (c) or (d) for the preparation of a kit for the detection of a novel coronavirus E484 variant;
(a) a specific probe as described in claim 6;
(b) a specific probe as described in claim 6 and a universal probe as described in claim 8;
(c) a specific probe as described in claim 6 and a specific primer pair as described in claim 7;
(d) the specific probe as claimed in claim 6 and the universal probe as claimed in claim 8 and the specific primer pair as claimed in claim 7.
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