CN114410848A - Composition, kit, method and use for detecting SARS-CoV-2 - Google Patents
Composition, kit, method and use for detecting SARS-CoV-2 Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention belongs to the field of molecular biology detection; in particular, it relates to the detection of SARS-CoV-2; more particularly, it relates to compositions, kits, methods and uses thereof for detecting SARS-CoV-2. The invention provides a kit comprising the composition, application of the composition, and a method for detecting and typing SARS-CoV-2 variant strains. Typing Ormcken and Deltay variant strains by detecting different characteristic functional variant sites, thereby simultaneously realizing the typing detection of SARS-CoV-2 variant strains in a single-tube reaction system. So that different strains can be treated differently, thereby making treatment and prevention more efficient. The composition has low cost, high flux and simple and convenient operation, and the result reading process can be judged through the Ct value of the amplification curve. The whole detection process is carried out under the condition of single tube sealing, so that false positive and environmental pollution are avoided.
Description
Technical Field
The invention belongs to the field of molecular biology detection; in particular, it relates to the detection of SARS-CoV-2; more particularly, it relates to the detection of the variant strains Ormckhen (Omicron) BA.1, BA.2 and Delta (Delta) of SARS-CoV-2.
Background
A novel coronavirus (2019-nCoV, SARS-CoV-2) was named SARS-CoV-2 by the International Committee for Classification of viruses at 11/2/2020. Coronaviruses are a large family of viruses, previously known to infect humans, such as those causing the common cold and Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS), while SARS-CoV-2 is a new strain of Coronavirus that has never been previously found in humans, a beta-type Coronavirus of the genus coronaviruses (Coronaviridae) in the family Coronaviridae (Coronavirus), an RNA ((+) ssRNA) virus with a capsule and spike cytological signature genome of a linear single positive strand.
According to the world health organization official network, hundreds of new variants of coronavirus have been discovered all over the world, and the most recent Onckrozen variants BA.1 and BA.2 have some important amino acid mutation sites of the first 4 variants needing attention, including sites with enhanced cell receptor affinity, virus replication capacity and immune escape capacity. Superposition of mutations may reduce the protective efficacy of some antibody drugs against the ormikosn variant. According to the study in UK and Denmark, BA.2 is more contagious than the original Omicron variant BA.1 by about 30%, and is more likely to cause serious disease, BA.2 requires closer attention, and BA.1 also has a spread rate increased by more than 70% compared with Delta strain, in addition to which the previously prevalent Delta still requires great attention. However, no reagent exists in the prior art for detecting Ormcken and Deltay variants and for distinguishing BA.1 from BA.2.
Therefore, there is a need in the art for reagents that can accurately detect the Ormcken variant and the Deltay variant in order to specifically take epidemic prevention and treatment measures to make the response more efficient. Meanwhile, the detection time is short, and the sensitivity is high.
Disclosure of Invention
In view of the above, in a first aspect, the present invention provides a composition capable of detecting and typing a SARS-CoV-2 variant strain, the composition comprising:
a first nucleic acid composition:
an upstream primer of the mutation T478K shown as SEQ ID NO. 1, a downstream primer of the mutation T478K shown as SEQ ID NO. 2, and a mutation T478K probe shown as SEQ ID NO. 3;
a second nucleic acid composition:
the upstream primer of the mutation HV69-70del shown as SEQ ID NO. 4, the downstream primer of the mutation HV69-70del shown as SEQ ID NO. 5, and the probe of the mutation HV69-70del shown as SEQ ID NO. 6;
a third nucleic acid composition:
an upstream primer of the mutation Q954H shown as SEQ ID NO. 7, a downstream primer of the mutation Q954H shown as SEQ ID NO. 8, and a mutation Q954H probe shown as SEQ ID NO. 9; and
a fourth nucleic acid composition:
the upstream primer of the mutation L452R shown in SEQ ID NO. 10, the downstream primer of the mutation L452R shown in SEQ ID NO. 11, and the mutation L452R probe shown in SEQ ID NO. 12.
The kit for detecting and typing SARS-CoV-2 variant strain provided by the invention mainly utilizes a multiple fluorescence PCR analysis method to type the Oncokrong variant strain and the Deltay variant strain by detecting 4 different characteristic function variant sites on the S gene of the SARS-CoV-2 variant strain, thereby simultaneously realizing the detection of the typing of the SARS-CoV-2 Oncokrong strains BA.1, BA.2 and the Deltay variant strain in a single-tube reaction system. So that different strains can be treated differently, thereby making treatment and prevention more efficient. The composition of the invention, combined with a fluorescent probe method, has low cost and high flux. And the operation is simple and convenient, and the result reading process can be judged through the Ct value of the amplification curve. The whole detection process is carried out under the condition of single tube sealing, so that false positive and environmental pollution caused by cross among samples are avoided.
Further, in some embodiments, the compositions of the invention may include one or more of the above-described primer and probe pairs simultaneously. In the present invention, "pair" refers to the matched upstream and downstream primers and probes for detecting a mutation.
For example, only the first nucleic acid composition may be included; may include only the second nucleic acid composition; may include only the third nucleic acid composition; only the fourth nucleic acid composition may be included.
Further, the fluorophores of the probes between the first, second, third and fourth nucleic acid compositions are different from each other and do not interfere with each other.
As used herein, "different from each other and non-interfering" means that the fluorophores used in each probe in the composition are different and do not interfere with each other's detection, i.e., detection can be performed using different channels. For example, FAM, HEX, ROX and CY5 can be used, which do not have close absorbance values and can select different channels and thus do not interfere with each other.
In a specific embodiment, the fluorescent reporter group of SEQ ID NO 3 is FAM; the fluorescent reporter group of SEQ ID NO 6 is VIC; the fluorescent reporter group of SEQ ID NO 9 is ROX; the fluorescent reporter group of SEQ ID NO. 12 is CY 5.
Further, the composition includes a universal primer.
In a particular embodiment, the composition further comprises: a universal upstream primer shown as SEQ ID NO. 13 and a universal downstream primer shown as SEQ ID NO. 14.
"including a universal primer" means that a universal primer is attached to the 5' end of each primer fragment, e.g., SEQ ID NO:1 connecting the universal primer sequences becomes:
TCTGTCCGTCTTCGCTTCAACCTTTTGAGAGAGATATTTCAACT。
the sequence of the SEQ ID NO 2 ligation universal primer is changed into:
TTCCGCTCACTCGTCACC CCATATGATTGTAAAGGAAAGTAACA。
by introducing the universal primer sequence fragments, different targets can be amplified by using the same universal primer pairs and a large number of target sequences are enriched, the occurrence of primer dimers can be reduced, and the competitive relationship between multiple specific amplification primer pairs is balanced, so that the amplification efficiency of the multiple PCR is obviously improved.
In some specific embodiments, the compositions of the invention are used in fluorescence PCR.
In the present invention, the fluorescent reporter group may be selected from FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3 and JOE, but is not limited thereto.
In a specific embodiment, the fluorescent reporter of the first nucleic acid composition probe is FAM; the fluorescent reporter group of the second nucleic acid composition probe is VIC; the fluorescent reporter of the third nucleic acid composition probe is ROX; and the fluorescent reporter of the fourth nucleic acid composition probe is CY 5.
Further, the 3' -end of the probe also has a quencher group, such as BHQ1 or BHQ2 or MGB.
In a specific embodiment, the 3' end of the probe is BHQ 1.
Further, the dosage of the primer in the composition is 0.1-0.3 mu M; the dosage of the probe in the composition is 0.1-0.3 mu M.
Furthermore, the dosage of the universal upstream primer in the composition is 0.5-0.8 mu M, and the dosage of the universal downstream primer is 0.6-1.0 mu M.
In a specific embodiment, each nucleic acid composition of the compositions of the invention is present in a separate package.
In a specific embodiment, each nucleic acid composition of the compositions of the invention is present in the same package.
Further, the components of each nucleic acid composition of the present invention are present in a mixed form.
In a second aspect, the present invention provides the use of the above-described composition of the present invention in the preparation of a kit for detecting and typing a SARS-CoV-2 variant.
In a third aspect, the present invention provides a kit for detecting and typing a SARS-CoV-2 variant, which comprises the composition of the present invention as described above.
Further, the kit also comprises a negative quality control product and a positive quality control product.
In a specific embodiment, the negative quality control is DEPC H2O, normal saline and reference gene pseudovirus. The positive quality control product is at least one of target fragment plasmid, fragment RNA and pseudovirus containing each mutation site of the novel coronavirus.
Further, the kit also comprises dNTP, PCR buffer solution and Mg2+At least one of (1).
Still further, the kit further comprises: at least one of a nucleic acid releasing agent, a nucleic acid extraction reagent, a reverse transcriptase, a uracil glycosylase, and a DNA polymerase.
Furthermore, the kit also comprises a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, reverse transcriptase, uracil glycosylase, DNA polymerase, PCR buffer solution and Mg2+At least one of (1).
Further, the concentration of the reverse transcriptase is 5U/reaction-15U/reaction, for example, the reverse transcriptase can be murine leukemia reverse transcriptase (MMLV) or Tth enzyme; the concentration of the DNA polymerase is 3U/reaction-15U/reaction, for example, the DNA polymerase can be Taq enzyme.
In a particular embodiment, the kit of the invention comprises: reverse/reverse transcriptase, Taq enzyme, uracil glycosylase, Mg2+、Mn2+Rnasin, dNTP, primers, probes and PCR buffer solution.
Common PCR buffers are Tris-HCl, MgCl2And buffer systems such as KCl and Triton X-100. The total volume of a single PCR reaction tube is 20-100 mu l generally.
In a specific embodiment, the kit of the present invention is compatible with a digital PCR amplification system, i.e., can be directly used for amplification on a digital PCR instrument.
In a fourth aspect, there is provided a method for detecting and typing a SARS-CoV-2 variant, the method comprising the steps of:
1) extracting or releasing nucleic acid of a sample to be detected;
2) performing a fluorescent quantitative PCR analysis on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) results were obtained and analyzed.
In the present invention, the sample to be detected may be a pharyngeal swab, an oropharyngeal swab, a nasopharyngeal swab, sputum, alveolar lavage fluid, blood, or the like, but is not limited thereto.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
reverse transcription is carried out at the temperature of 50-60 ℃ for 5-30 minutes, and 1 cycle is carried out; pre-denaturing cDNA at 95 deg.c for 1-10 min for 1 circulation; and (3) denaturation at 95 ℃ for 5-20 seconds, annealing at 55-60 ℃ for 20-60 seconds, and collecting fluorescence after 40-50 cycles.
In a specific embodiment, a method is provided for detecting and typing a SARS-CoV-2 variant strain for non-diagnostic purposes, the method comprising the steps of:
1) extracting or releasing nucleic acid of a sample to be detected;
2) performing a fluorescent quantitative PCR analysis on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) results were obtained and analyzed.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
reverse transcription is carried out at the temperature of 50-60 ℃ for 5-30 minutes, and 1 cycle is carried out; pre-denaturing cDNA at 95 deg.c for 1-10 min for 1 circulation; and (3) denaturation at 95 ℃ for 5-20 seconds, annealing at 55-60 ℃ for 20-60 seconds, and collecting fluorescence after 40-50 cycles.
As used herein, the term "non-diagnostic purpose" refers to information that is not intended to obtain information on whether an individual is infected with a variant of SARS-CoV-2 Onckhun and has pneumonia. For example, the method can be used to detect the presence or absence of SARS-CoV-2 variant in a test culture in an experiment for research purposes and to type the same.
Drawings
FIG. 1 shows the results of the test of Ormcken variant strain BA.1 by the composition of the present invention;
FIG. 2 shows the results of the test of Ormcken variant strain BA.2 by the composition of the present invention;
FIG. 3 shows the results of detecting a delta variant with the composition of the present invention;
FIG. 4 shows the results of the sensitivity test of the composition of the present invention for detecting Ormcken variant strain BA.1 (100 copies/mL);
FIG. 5 shows the results of the sensitivity test of the composition of the present invention for detecting Ormcken variant strain BA.2 (100 copies/mL);
FIG. 6 shows the results of the sensitivity test (100 copies/mL) of the composition of the present invention for detecting a delta variant;
FIG. 7 shows the specific detection of the composition of the present invention (human coronavirus HKU 1/human coronavirus OC 43);
FIG. 8 shows the specific detection of a composition of the invention (human coronavirus NL 63/human coronavirus 229E);
FIG. 9 shows the specific detection of the composition of the present invention (SARS coronavirus/MERS coronavirus);
FIG. 10 shows the specific detection of the composition of the present invention (influenza A virus/influenza B virus);
FIG. 11 shows the specific detection of the novel crown variant of the composition of the present invention (negative sample and other variant samples);
FIG. 12 shows the specific detection of a novel crown variant (wild-type sample) according to the composition of the present invention;
FIG. 13 shows the results of the test of comparative example 1, composition 1 according to the present invention;
FIG. 14 shows the results of the test of comparative example 1, composition 2 according to the present invention;
FIG. 15 shows the results of the test of comparative example 1, composition 3 according to the present invention.
Detailed Description
In the present invention, the expressions "first", "second", "third", and "fourth", etc. are used for descriptive purposes only to distinguish between the defined substances, and not to define an order or primary or secondary in any way.
The present invention will be specifically explained below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are illustrative of the invention and are not to be construed as limiting the invention.
Example 1 primers and probes used in the present invention
TABLE 1
Wherein, the fluorescent reporter group of SEQ ID NO. 3 is FAM; the fluorescent reporter group of SEQ ID NO 6 is VIC; the fluorescent reporter group of SEQ ID NO 9 is ROX; the fluorescent reporter group of SEQ ID NO. 12 is CY 5.
Example 2 method for detecting SARS-CoV-2
Specimen type: oropharyngeal swab, nasopharyngeal swab.
Nucleic acid extraction:
a commercial RNA extraction kit, such as a nucleic acid extraction reagent based on a silica gel membrane centrifugal column method or a nucleic acid extraction reagent based on a magnetic bead method, is adopted, the operation is carried out according to the kit instruction, and finally 80 mu L of RNA solution is collected and directly detected.
Or stored at-80 ℃. The negative quality control product and the positive quality control product are extracted.
System configuration:
according to the total reaction number N required for detection, 18.5. mu.l of RT-PCR amplification solution (MgCl) is added into each tube of PCR26mM, dNTP 0.8mM, and the concentration of the forward primer/the backward primer were all 0.2. mu.M, the concentration of the probe was 0.25. mu.M, the concentration of the universal forward primer was 0.6. mu.M, and the concentration of the universal backward primer was 0.6. mu.M), and 1.5. mu.l of an enzyme mixture (hot-start Taq enzyme, reverse transcriptase, uracil glycosylase (UNG)). Calculating the required total amount, mixing uniformly and then packaging into a special PCR reaction tube. Sample adding:
adding a negative quality control product, a sample RNA solution and a positive quality control product into the PCR reaction tube which is filled with the reagents respectively, wherein the adding amount of the sample RNA solution and the positive quality control product is 10 mu L, tightly covering a tube cover, uniformly mixing, centrifuging, collecting the solution, and placing the solution at the bottom of the tube.
Performing on-machine amplification detection:
the settings of the RT-PCR amplification program and the melting curve analysis program are shown in Table 2 below:
TABLE 2
And (4) analyzing results:
on the premise that the amplification is effective, the judgment is as shown in table 3:
TABLE 3
HV69-70del amplification curve positive [ Ct (+) ] indicates no HV69-70del mutation carried in the sample, and HV69-70del amplification curve negative [ Ct (-) ] indicates HV69-70del mutation carried in the sample.
The typing criteria of each variant of the novel coronavirus are shown in Table 4:
TABLE 4
Example 3 test results of test specimens of the composition of the invention
The primers and probes shown in example 1 were used to detect the pseudoviruses of Ormcken and Deltay according to the method of example 2, and the results are shown in FIGS. 1 to 3. The results show that each channel can be detected normally, and the multiplex PCR system can detect the condition of the corresponding target and classify the Ormcken and Deltay.
Example 4 sensitivity of the compositions of the invention
Omicron BA.1, BA.2 and Delta new crown variant pseudoviruses are respectively diluted to 100 copies/mL by negative samples to verify the sensitivity of the reagent and the detection method. The detection results are shown in FIGS. 4-6, and the pseudovirus simulated samples with 100 copies/mL of Omicron BA.1, BA.2 and Delta can be accurately detected, and the typing is correct, which indicates that the detection method has higher sensitivity.
Example 5 specificity of the compositions of the invention
Pseudoviruses of endemic human coronavirus (HKU 1, OC43, NL63 and 229E), SARS coronavirus, MERS coronavirus, influenza A virus, and influenza B virus were diluted to 1X 106When the copy/mL is used as a specificity detection sample and detected by using the composition disclosed by the embodiment 1 of the invention, no specific amplification occurs in an experimental result, and partial detection results are shown in FIGS. 7-10.
The detection results show that 8 pathogens including endemic human coronavirus (HKU 1, OC43, NL63 and 229E), SARS coronavirus, MERS coronavirus, influenza A virus and influenza B virus are negative, and the composition has good specificity.
Example 6 specificity of novel crown variants of the compositions of the invention
In order to verify the accuracy and effectiveness of differential diagnosis of the Omicron BA.1, BA.2 and Delta typing detection kit and other variants of the new corona virus, negative samples and pseudovirus samples of Alpha, Beta, Gamma, Lambda and Mu of the other variants of the new corona virus are verified, and the results of the Omicron BA.1, BA.2 and Delta typing detection kit show that the Omicron BA.1, BA.2 and Delta are typed correctly and do not have cross reaction with the other variants, which indicates that the kit can accurately perform differential diagnosis on the Omicron BA.1, BA.2 and Delta variants and other variants, and the detection is shown in figures 11-12.
Comparative example 1 primers and probes designed according to the invention with the remaining Effect not good
Because of the base complementary pairing principle, a dimer is formed between the primer and (or) the probe, but the probability is very small, and the dimer can be excluded at the beginning of the design. However, when multiple pathogens are jointly detected, a large number of primers and probes exist, dimers are easy to occur between the primers and the primers, between the probes and the probes, the designed conservativeness is ensured (the conservativeness is important for the detection accuracy), and the mutual interference between different primer probes is considered, so that the primer probes need to be designed elaborately.
Therefore, the inventors also designed the remaining primers and probes (sequences not shown) to constitute different detection systems 1, 2 and 3 (comparative example 1, compositions 1, 2 and 3), and also used for detection of variants. The specific detection results are shown in fig. 13-15, and it can be seen from the graphs that the fluorescence amplification of the detection amplification curve is low, the detection effect of the sensitivity is poor, and part of targets even have no amplification curve, so that the overall detection effect is poor.
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Claims (10)
1. A composition capable of detecting and typing a SARS-CoV-2 variant, said composition comprising in combination:
a first nucleic acid composition:
an upstream primer of the mutation T478K shown as SEQ ID NO. 1, a downstream primer of the mutation T478K shown as SEQ ID NO. 2, and a mutation T478K probe shown as SEQ ID NO. 3;
a second nucleic acid composition:
the upstream primer of the mutation HV69-70del shown as SEQ ID NO. 4, the downstream primer of the mutation HV69-70del shown as SEQ ID NO. 5, and the probe of the mutation HV69-70del shown as SEQ ID NO. 6;
a third nucleic acid composition:
an upstream primer of the mutation Q954H shown as SEQ ID NO. 7, a downstream primer of the mutation Q954H shown as SEQ ID NO. 8, and a mutation Q954H probe shown as SEQ ID NO. 9; and
a fourth nucleic acid composition:
the upstream primer of the mutation L452R shown in SEQ ID NO. 10, the downstream primer of the mutation L452R shown in SEQ ID NO. 11, and the mutation L452R probe shown in SEQ ID NO. 12.
2. The composition of claim 1, wherein the fluorophores of the probes between the first, second, third, and fourth nucleic acid compositions are different from each other and do not interfere with each other.
3. The composition of claim 2, wherein the fluorescent reporter of SEQ ID NO:3 is FAM; the fluorescent reporter group of SEQ ID NO 6 is VIC; the fluorescent reporter group of SEQ ID NO 9 is ROX; the fluorescent reporter group of SEQ ID NO. 12 is CY 5.
4. The composition of claim 1, wherein the composition comprises a universal primer.
5. The composition of claim 4, wherein the composition further comprises: a universal upstream primer shown as SEQ ID NO. 13 and a universal downstream primer shown as SEQ ID NO. 14.
6. The composition of any one of claims 1 to 5, wherein each nucleic acid composition of the composition is present in the same package.
7. Use of the composition of any one of claims 1 to 6 in the preparation of a kit for detecting and typing a SARS-CoV-2 variant.
8. A kit for detecting and typing a SARS-CoV-2 variant, the kit comprising the composition of any one of claims 1 to 6.
9. The kit of claim 8, wherein the kit further comprises a nucleic acid releasing reagent, a nucleic acid extracting reagent, dntps, reverse transcriptase, uracil glycosylase, DNA polymerase, PCR buffer, and Mg2+At least one of (1).
10. A method for detecting and typing a SARS-CoV-2 variant for non-diagnostic purposes, the method comprising the steps of:
1) extracting or releasing nucleic acid of a sample to be detected;
2) performing fluorescence quantitative PCR and melting curve analysis on the nucleic acid obtained in the step 1) by using the composition according to any one of claims 1 to 6 or the kit according to any one of claims 8 to 9;
3) results were obtained and analyzed.
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Cited By (2)
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| CN114592097A (en) * | 2022-05-07 | 2022-06-07 | 北京生物制品研究所有限责任公司 | Primer and probe for identifying novel coronavirus Omicron strain BA.1 and/or BA.3 sublines and application thereof |
| CN117344062A (en) * | 2023-12-06 | 2024-01-05 | 江苏硕世生物科技股份有限公司 | Kit for rapidly detecting novel coronavirus nucleic acid and application method |
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