CN114561490B - Composition, kit and method for detecting SARS-CoV-2 mutation site and application thereof - Google Patents
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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 specifically, it relates to the detection of the major mutation site of SARS-CoV-2 mutant. Meanwhile, a kit comprising the composition, the use of the composition, and a method for detecting and typing a SARS-CoV-2 mutant strain are also provided. The composition of the invention can be used for identifying main mutations on alpha mutant strains, beta mutant strains, gamma mutant strains and Ormcken mutant strains, and carrying out preliminary screening and confirmation on the mutant strain types through site combination detection, so that different mutation sites can be treated differently, and the treatment and prevention are more efficient. The composition of the invention combines a fluorescent probe method to detect 5 targets simultaneously, and has the advantages of low cost, high flux, simple and convenient operation and short time.
Description
Cross Reference to Related Applications
The present application claims priority from chinese patent application having application date of 09/12/2021, application No. 202111500433.X entitled "composition, kit, method and use for detecting a mutation site of SARS-CoV-2", the entire contents of which are incorporated herein by reference.
Technical Field
The invention belongs to the field of molecular biology detection; in particular, it relates to the detection of SARS-CoV-2; more specifically, it relates to the detection of the major mutation site of SARS-CoV-2 mutant strain.
Background
One of the biggest characteristics of the new coronavirus, which is an RNA virus, is easy mutation, and the new coronavirus carries stronger transmission power each time from a variant strain Alpha (Alpha B.1.1.7), beta (Beta B.1.351), gamma (Gamma P.1) and Delta (Delta B.1.617.2).
SARS-CoV-2 is more susceptible to deletion (mutations) that occur most frequently in Repetitive Deletion Regions (RDR) of the S protein. The SARS-CoV-2 mutant strain also occurs in succession in many parts of the world. In the last few months, several remarkable SARS-CoV-2 mutants appeared, such as the b.1.1.7 mutant in uk, the p.1 mutant in brazil and the b.1.351 mutant in south africa (also known as 501y.v 2). B.1.1.7 mutants appeared in uk 12 months in 2020, and were then disseminated into more than a dozen countries; its genome has 17 mutations, 8 of which are in the S protein, and the transmission ability is significantly enhanced. The P.1 mutant began to be prevalent in Brazil in mid 2020. The b.1.351 mutant appeared in south africa at 12 months 2020. The common feature of these 3 mutants is that multiple mutations occur in the S protein and rapidly evolve into dominant epidemic strains. Deletion of RDRs as an antibody recognition epitope may cause the binding specificity of SARS-CoV-2 antibody to change, so that antigen evolution (antigenic evolution) occurs, and then a neutralizing antibody induced by SARS-CoV-2 before mutation escapes. The occurrence of the mutant strain also has great influence on epidemic situation in China. It was reported that 1 case of British B.1.1.7 subtype was found in Guangdong province at 1/2/2021, and the viral genome sequence contained 13 branch-specific amino acid variation sites of mutant B.1.1.7 subtype strains, including N501Y, P681H mutation of S protein, which has a potentially important influence on the ability of the virus to infect, and 69-70 deletion mutation of S protein associated with viral immune escape. 1 example of a 501y.v2 south african mutant was found in Guangdong province at 1/6, except that the same N501Y mutation as the British mutant B.1.1.7 subtype was included in the mutations at two key sites of S proteins E484K and K417N, which potentially have a significant effect on the ability to infect viruses. The B.1.525 Nigeria mutant was found again at 12 days 3 months. The world health organization announced in 26 days 11.11.1 that south africa discovered a new crown variant strain and rapidly listed as the highest level "high concern variant" named Omi-cron (Greek letter designation), and Chinese translated as "Oncakjon". These mutants are rapidly spreading and gradually replace other types of new coronaviruses that are spread. The prevention and control of epidemic situations face huge challenges, and the development of vaccines is urgent. Some mutant strains may generate resistance to antibodies induced by the existing SARS-CoV-2 vaccine, and influence the effects of detection reagents and vaccines, so that the determination of the mutation sites of the virus has great significance for epidemiological analysis and clinical diagnosis and treatment of the virus.
There is a need in the art for an agent that can accurately identify the mutation sites of different mutants of neocorolla and preliminarily identify the type of mutant so that epidemic prevention and treatment measures can be taken with a high degree of efficiency. 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 the major mutation site of SARS-CoV-2 mutant strain and determining and identifying the mutant strain type by combining the detection results of different sites, the composition comprising:
a first nucleic acid composition:
a mutation N501Y upstream primer shown as SEQ ID NO. 1, a mutation N501Y downstream primer shown as SEQ ID NO. 2, and a mutation N501Y probe shown as SEQ ID NO. 3;
a mutant HV69-70del upstream primer as shown in SEQ ID NO. 4, a mutant HV69-70del downstream primer as shown in SEQ ID NO. 5, and a mutant HV69-70del probe as shown in SEQ ID NO. 6; and
a second nucleic acid composition:
a mutation K417N upstream primer shown as SEQ ID NO. 7, a mutation K417N downstream primer shown as SEQ ID NO. 8 and a mutation K417N probe shown as SEQ ID NO. 9;
a mutation E484K upstream primer shown as SEQ ID NO. 10, a mutation E484K downstream primer shown as SEQ ID NO. 11, and a mutation E484K probe shown as SEQ ID NO. 12;
a mutation P681H upstream primer shown as SEQ ID NO. 13, a mutation P681H downstream primer shown as SEQ ID NO. 14, and a mutation P681H probe shown as SEQ ID NO. 15.
The composition can be used for identifying main mutations on alpha mutant strains, beta mutant strains, gamma mutant strains and Ormcken mutant strains, and judging and identifying the mutant strain types through the combination of detection results of different sites, so that different mutation types can be treated differently, and the treatment and prevention are more efficient. The composition of the invention combines a fluorescent probe method to detect 5 targets simultaneously, and has the advantages of low cost, high flux, simple and convenient operation and short time.
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 matched upstream and downstream primers and probes for detecting a mutation.
For example, only the first nucleic acid composition may be included; only the second nucleic acid composition may be included.
For example, any combination of primer and probe pairs within different nucleic acid compositions may be included, for example, including only the mutant N501Y upstream primer shown in SEQ ID NO. 1, the mutant N501Y downstream primer shown in SEQ ID NO. 2, and the mutant N501Y probe shown in SEQ ID NO. 3, the mutant K417N upstream primer shown in SEQ ID NO. 7, the mutant K417N downstream primer shown in SEQ ID NO. 8, and the mutant K417N probe shown in SEQ ID NO. 9.
Further, the fluorophores of the probes within each of the first and second 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., they can be detected 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.
Further, the composition comprises: an internal standard upstream primer, an internal standard downstream primer and an internal standard probe for monitoring.
Further, the internal standard comprises a human genome internal standard.
In a particular embodiment, the composition further comprises: an upstream primer of the human genome internal standard shown as SEQ ID NO. 16, a downstream primer of the human genome internal standard shown as SEQ ID NO. 17 and a probe of the human genome internal standard shown as SEQ ID NO. 18.
Further, the internal standard comprises a new corona internal standard.
In a particular embodiment, the composition further comprises: a new crown internal standard upstream primer shown as SEQ ID NO. 19, a new crown internal standard downstream primer shown as SEQ ID NO. 20 and a new crown internal standard probe shown as SEQ ID NO. 21.
Further, the internal standards include a human genome internal standard and a new corona internal standard.
In a particular embodiment, the composition further comprises: a human genome internal standard upstream primer shown as SEQ ID NO. 16, a human genome internal standard downstream primer shown as SEQ ID NO. 17 and a human genome internal standard probe shown as SEQ ID NO. 18; and a new crown internal standard upstream primer shown as SEQ ID NO. 19, a new crown internal standard downstream primer shown as SEQ ID NO. 20 and a new crown internal standard probe shown as SEQ ID NO. 21.
Further, the internal standard probe and the fluorescent group of the probe within each set of the first and second nucleic acid compositions are different from each other and do not interfere with each other.
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.
Further, the 3' -end of the probe also has a quenching group, such as BHQ1 or BHQ2.
In a specific embodiment, the 3' end of the probe is BHQ1.
Furthermore, the dosage of the primer in the composition is 0.1-0.3 mu M; the dosage of the probe in the composition is 0.15-0.25 mu M.
In a particular embodiment, each nucleic acid composition of the invention is present in a separate 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 the major mutation site of SARS-CoV-2 mutant strain.
In a third aspect, the present invention provides a kit for detecting the major mutation site of SARS-CoV-2 mutant strain, 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 H 2 O, normal saline and reference gene pseudovirus. Positive quality controlThe product contains at least one of new coronavirus ORF 1ab target gene, new coronavirus N target gene, new coronavirus mutation sites, target fragment plasmid of reference gene, fragment RNA and pseudovirus.
Further, the kit further comprises: a nucleic acid delivery system and a nucleic acid amplification system.
Further, the kit also comprises dNTP, PCR buffer solution and Mg 2+ 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 Mg 2+ At least one of (a).
Further, the concentration of the reverse transcriptase is 5U/reaction to 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 to 15U/reaction, for example, the DNA polymerase may be Taq enzyme.
In a particular embodiment, the kit of the invention comprises: reverse/reverse transcriptase, taq enzyme, uracil glycosylase, mg 2+ 、Mn 2+ Rnasin, dNTP, primers, probes and PCR buffer solution.
Common PCR buffers are Tris-HCl, mgCl 2 And buffer systems such as KCl and Triton X-100. The total volume of a single PCR reaction tube is generally 20 to 100. Mu.l.
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 a major mutation site of a SARS-CoV-2 mutant strain, 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 used for detection may be a pharyngeal swab, an oropharyngeal swab, a nasopharyngeal swab, sputum, alveolar lavage fluid, blood, and 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 circulation is carried out; pre-denaturing cDNA at 95 deg.c for 1-10 min for 1 circulation; denaturation at 95 deg.C for 5-20 s, annealing at 55-60 deg.C for 20-60 s, and collecting fluorescence after 40-50 cycles.
In a specific embodiment, the reaction conditions of the fluorescent quantitative PCR are: reverse transcription at 50 deg.c for 10 min for 1 circulation; pre-denaturation of cDNA at 95 deg.C for 1 min for 1 cycle; denaturation at 95 ℃ for 10 seconds, annealing at 60 ℃ for 20 seconds, and collecting fluorescence after 45 cycles.
In a specific embodiment, a method is provided for detecting the major mutation site of a SARS-CoV-2 mutant 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 at 50-60 deg.c for 5-30 min for 1 circulation; pre-denaturation of cDNA at 95 deg.c for 1-10 min for 1 circulation; denaturation at 95 deg.C for 5-20 s, annealing at 55-60 deg.C for 20-60 s, and collecting fluorescence after 40-50 cycles.
In a specific embodiment, the reaction conditions of the fluorescent quantitative PCR are: reverse transcription at 50 deg.c for 10 min for 1 circulation; pre-denaturation of cDNA at 95 deg.C for 1 min for 1 cycle; denaturation at 95 ℃ for 10 seconds, annealing at 60 ℃ for 20 seconds, and collecting fluorescence after 45 cycles.
As used herein, the term "non-diagnostic purpose" refers to information that is not intended to indicate whether an individual is infected with the SARS-CoV-2 mutant strain and has suffered pneumonia. For example, the method can be used to detect the presence of SARS-CoV-2 mutant in the test culture in experiments aimed at research.
Drawings
FIGS. 1 to 4 show the results of measurements on samples tested with the composition of the present invention (N501Y and HV69-70del, P681H, E484K, K417N, respectively);
FIGS. 5 to 7 are the results of detecting samples (E484K, K417N, P681H, respectively) with different concentrations by using the composition of the present invention;
FIG. 8 shows the result of specific detection of the composition of the present invention;
FIGS. 9 to 10 show results of measurement of precision (E484K, K417N, respectively) of the composition of the present invention;
FIGS. 11 to 14 show the results of measurement of comparative example compositions of the present invention (E484K, K417N, P681H, HV69 to 70del, respectively).
Detailed Description
In the present invention, the expressions "first" and "second" 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
The primers and probes used in the present invention are shown in table 1 below:
TABLE 1
Wherein, the fluorescent groups of N501Y-P and E484K-P are FAM; the fluorescent groups of HV69-70Del-P and K417N-P are HEX; the fluorescent groups of P681H-P and IC-P are CY5; the fluorophore of N-P is ROX.
Example 2 method for detecting novel mutant strains of coronavirus
The detection sample of the invention is throat swab, sputum, alveolar lavage fluid and blood. The magnetic bead method is used for extracting virus nucleic acid (Shengxiang biological products, inc. kit), and the following operations are carried out in a sample processing chamber:
2.1 taking a proper amount of 1.5mL of sterilized centrifuge tubes, respectively marking a negative control, a positive control and a sample to be detected, and adding 300 mu L of RNA extraction solution 1 into each tube;
2.2 adding 200 mul sample to be tested or negative control and positive control into each tube; covering a tube cover, shaking and uniformly mixing for 10 seconds, and instantaneously centrifuging;
2.3 adding 100 mul of RNA extraction solution 2-mix (sucking after fully mixing) into each tube, shaking and mixing for 10 seconds, and standing for 10 minutes at room temperature;
2.4 placing the centrifuge tube on a separator after instantaneous centrifugation, slowly sucking out the solution after 3 minutes (take care not to touch brown adsorbed on the tube wall);
2.5 adding 600 mul of RNA extraction solution 3 and 200 mul of RNA extraction solution 4 into each tube, shaking and uniformly mixing for 5 seconds, and placing the centrifugal tube on the separator again after instantaneous centrifugation;
after 2.6 minutes and about 3 minutes, separating the supernatant into two layers, inserting a suction head into the bottom of a centrifuge tube, slowly and completely sucking out and discarding the liquid from the bottom, and standing for 1 minute, and completely sucking out and discarding the residual liquid at the bottom of the centrifuge tube;
2.7 mu.L of PCR-mix was added to each tube, the brown residue adsorbed on the wall of the centrifuge tube was eluted by sucking the PCR-mix with a tip, and the elution was repeated several times as completely as possible, and then the whole brown mixture after elution was transferred to a 0.2mL PCR reaction tube, and the tube was covered and transferred to an amplification detection zone.
The real-time fluorescent PCR reaction system is configured as follows:
the PCR amplification program was set up as follows:
and (4) analyzing results:
1) The target detection signal is FAM, HEX (or VIC) and ROX, and the internal reference detection signal is CY5/ROX;
2) Setting Baseline: baseline is generally set to be 3-15 cycles, and can be adjusted according to actual conditions. The adjustment principle is as follows: selecting a region with stable fluorescence signal before exponential amplification, wherein the starting point (Start) avoids the signal fluctuation in the initial stage of fluorescence acquisition, and the End point (End) is reduced by 1-2 cycles compared with the sample Ct with the earliest exponential amplification. Setting Threshold: setting a rule that a threshold value line just exceeds the highest point of a normal negative control product;
3) And (4) interpretation of results:
the invention can realize rapid and accurate preliminary screening identification through combined interpretation of different sites. Meanwhile, the kit adopts the new crown conserved sequence N gene as a monitoring internal standard, so that the accuracy of a sample detection result is improved.
Example 3 test results of the inventive composition for testing Positive controls
Using the compositions of Table 1 of the present invention, fluorescence quantitative PCR was performed on clinical samples of the new corona mutation (positive) at the first hospital of Changsha, hunan, according to the method described in example 2, to detect several major mutation sites of the novel coronavirus nucleic acid, including mutation sites such as E484K, K417N, P681H, N501Y, and HV69-70del, one of which was found to have typical mutations such as N501Y, HV69-70del, and P681H (FIGS. 1-2), and one of which was found to have mutations such as E484 and K417N (FIGS. 3-4). As can be seen from the figures, the compositions of the invention can detect corresponding targets, and prove that the compositions of the invention can detect new coronavirus mutant strains.
Example 4 sensitivity test of compositions of the invention
The results of multiplex PCR detection using the compositions of Table 1 of the present invention and the method described in example 2 were shown in FIGS. 5 to 7, in which 5 concentrations of 200000, 20000, 2000, 200, and 20 copies/ml of each target pseudovirus were detected and the multiplex PCR detection was performed on a fluorescence quantitative PCR instrument using a Macro stone. As can be seen from the figure, the concentration as low as 200 copies/ml can still detect the corresponding target, demonstrating the composition sensitivity of 200 copies/ml.
Example 5 specificity assay of compositions of the invention
Using the compositions of Table 1 of the present invention, multiple PCR assays were performed on a quantitative PCR instrument using fluorescence from Macro-lithophane according to the method described in example 2 for pathogens (e.g., coronavirus (NL 63, HKU1, 229E, OC43), influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, parainfluenza virus, klebsiella pneumoniae, streptococcus pneumoniae, haemophilus influenzae, pseudomonas aeruginosa, legionella pneumophila, bordetella pertussis, staphylococcus aureus, mycoplasma pneumoniae, chlamydia pneumoniae, etc.) that have homology in nucleic acid sequence and are likely to cause the same or similar clinical symptoms, as shown in FIG. 8. As can be seen from the figure, the detection is negative, and only the internal standard gene of the human genome is amplified. The compositions of the invention proved to be very specific.
Example 6 precision testing of compositions of the invention
Using the compositions of Table 1 of the present invention, quality controls of 2 concentration levels of strong and weak positive (100000 and 2000 copies/ml, respectively) were selected for measurement of in-batch precision and inter-batch precision as described in example 2, and the measurement was repeated 10 times for each sample. The results show that the detection rates of the strong and weak positive reference products are both 100%, and the variation Coefficient (CV) of the detected Ct values in batches and between batches is less than 5%, as shown in FIGS. 9-10. The kit shows that the kit has good detection precision in batch and batch.
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 are provided, dimers are easily generated 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, and also used for detecting the new crown mutation. Specific detection results are shown in fig. 11 to 14, and it can be seen that the detection effect is poor.
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<212> DNA
<213> Artificial sequence
<400> 21
tgcaacccat atgatgccgt 20
Claims (7)
1. A composition capable of detecting the major mutation site of a SARS-CoV-2 mutant, said composition comprising, in combination:
a first nucleic acid composition:
a mutation N501Y upstream primer shown as SEQ ID NO. 1, a mutation N501Y downstream primer shown as SEQ ID NO. 2, and a mutation N501Y probe shown as SEQ ID NO. 3;
an upstream primer of the mutation HV69-70del shown as SEQ ID NO. 4, a downstream primer of the mutation HV69-70del shown as SEQ ID NO. 5, and a probe of the mutation HV69-70del shown as SEQ ID NO. 6;
a human genome internal standard upstream primer shown as SEQ ID NO. 16, a human genome internal standard downstream primer shown as SEQ ID NO. 17 and a human genome internal standard probe shown as SEQ ID NO. 18; and
a new crown interior label upstream primer shown as SEQ ID NO. 19, a new crown interior label downstream primer shown as SEQ ID NO. 20 and a new crown interior label probe shown as SEQ ID NO. 21; and
a second nucleic acid composition:
a mutation K417N upstream primer shown as SEQ ID NO. 7, a mutation K417N downstream primer shown as SEQ ID NO. 8 and a mutation K417N probe shown as SEQ ID NO. 9;
a mutation E484K upstream primer shown as SEQ ID NO. 10, a mutation E484K downstream primer shown as SEQ ID NO. 11, and a mutation E484K probe shown as SEQ ID NO. 12;
a mutation P681H upstream primer shown as SEQ ID NO. 13, a mutation P681H downstream primer shown as SEQ ID NO. 14, and a mutation P681H probe shown as SEQ ID NO. 15; and
a new crown internal standard upstream primer shown as SEQ ID NO. 19, a new crown internal standard downstream primer shown as SEQ ID NO. 20 and a new crown internal standard probe shown as SEQ ID NO. 21.
2. The composition of claim 1, wherein each nucleic acid composition of the composition is present in a separate package.
3. Use of the composition of claim 1 or 2 in the preparation of a kit for detecting the major mutation site of the SARS-CoV-2 mutant strain.
4. A kit for detecting the major mutation site of SARS-CoV-2 mutant strain, the kit comprising the composition of claim 1 or 2.
5. The kit of claim 4, wherein the kit further comprises a nucleic acid delivery system and a nucleic acid amplification system.
6. The kit of claim 5, 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 Mg 2+ At least one of (1).
7. A method for detecting the major mutation site of a SARS-CoV-2 mutant 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 claim 1 or 2;
3) Results were obtained and analyzed.
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| EP22735762.1A EP4232608A2 (en) | 2021-05-28 | 2022-05-24 | Composition, kit, method, and use thereof for detecting sars-cov-2 mutation sites |
| PCT/CN2022/094749 WO2022247833A2 (en) | 2021-05-28 | 2022-05-24 | Composition, kit, method, and use thereof for detecting sars-cov-2 mutation sites |
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| CN202111500433X | 2021-12-09 | ||
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