EP4232608A2 - Composition, kit, method, and use thereof for detecting sars-cov-2 mutation sites - Google Patents
Composition, kit, method, and use thereof for detecting sars-cov-2 mutation sitesInfo
- Publication number
- EP4232608A2 EP4232608A2 EP22735762.1A EP22735762A EP4232608A2 EP 4232608 A2 EP4232608 A2 EP 4232608A2 EP 22735762 A EP22735762 A EP 22735762A EP 4232608 A2 EP4232608 A2 EP 4232608A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- seq
- primer
- probe
- mutant
- composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Definitions
- the present application claims priority to: the application No. 202111500433. X, filed on December 09, 2021, entitled “Composition, Kit, Method and Use thereof for Detecting SARS-CoV-2 Mutation Sites” , the application No. 202110592489.6, filed on May 28, 2021, entitled “Combination Product, Kit, Use and Method for Detecting Mutant SARS-CoV-2 Viruses” , the application No. 202111503790.1, filed on December 10, 2021, entitled “Composition, Kit, Method and Use thereof for Detecting SARS-CoV-2 Mutation Sites” , and the application No.
- the present invention relates to the field of molecular biology detection, specifically to detection of SARS-CoV-2, and more specifically to detection of major mutation sites of SARS-CoV-2 variants.
- RNA virus As an RNA virus, one of the significant characteristics of the novel coronavirus is easy mutation, from variant alpha (Alpha B. 1.1.7) to beta (Beta B. 1.351) , again to gamma (Gamma P. 1) and delta (Delta B. 1.617.2) , after each mutation, the virus attains stronger virus transmissibility.
- the Delta variant found in India is becoming the most prevalent variant of the novel coronavirus in the world due to its significantly enhanced transmission ability.
- the new variant evades the line of defense of many countries, such as east Asian countries including China, and southeast Asian countries, and after breaking the line of defense of public health prevention and control, the variant becomes a challenge to the vaccine defensive line.
- mutations thereof mainly occur at 5 genes, i.e., S gene, N gene, ORF8 gene, ORF3a gene and ORF1ab gene, wherein the mutation on S gene has a greatest effect on the transmission and pathogenicity of the virus.
- the most prevalent variants mainly include B. 1.1.7, B. 1.351, B. 1.617.1, B.1.617.2, B. 1.617.3, B. 1.525, P. 1, P. 2, C. 37 and the like.
- Major mutation sites are E484Q, E484K, L452R, P681R, T487K, D950N, F490S, Del247-253, T76I, L452Q, and the like, and these sites may lead to increased immune escape or infectivity.
- Delta and Lambda variants said variants are spreading rapidly and are gradually superseding other types novel coronavirus.
- a huge challenge is generated for epidemic prevention and control, and development of vaccines is also urgent.
- Some of the mutations may be resistant to antibodies induced by current SARS-CoV-2 vaccines, and affect the efficacy of detection reagents and vaccines. Therefore, identifying mutation sites of the virus is of great significance to epidemiological analysis and clinical diagnosis of the virus, and treatment related thereto.
- a reagent is required in the art, which can be used to accurately identify different mutations of the novel coronavirus, so that targeted epidemic prevention and treatment measures can be taken to make the response more efficient. Moreover, the detection time must be short and the sensitivity must be high.
- the present invention provides a composition which can be used to detect major mutation sites of SARS-CoV-2 variants and identify the mutant types by combining detection results for different sites, the composition simultaneously comprising:
- a primer and probe combination for detecting mutation N501Y a primer and probe combination for detecting mutation P681H, and a primer and probe combination for detecting mutation HV69-70del;
- primer and probe combination for detecting mutation N501Y is selected from one or more of the following groups:
- primer and probe combination for detecting mutation P681H is selected from one or more of the following groups:
- the primer and probe combination for detecting mutation HV69-70del is selected from one or more of the following groups:
- composition of the present invention may simultaneously include one or more pairs of the primers and probes described above.
- “pair” refers to mutually matched upstream and reverse primers and probes for detecting mutations.
- the composition can include one, two, or three primer and probe combinations for detecting mutation N501Y.
- the composition can include one, two, or three primer and probe combinations for detecting mutation P681H.
- the composition can include one, two, or three primer and probe combinations for detecting mutation HV69-70del.
- the composition simultaneously comprises one of the primer and probe combinations for detecting mutation N501Y, one of the primer and probe combinations for detecting mutation P681H, and one of the primers and probes for detecting mutation HV69-70del.
- the composition simultaneously comprises two of the primer and probe combinations for detecting mutation N501Y, two of the primer and probe combinations for detecting mutation P681H, and two of primers and probes for detecting mutation HV69-70del.
- the composition simultaneously comprises three of the primer and probe combinations for detecting mutation N501Y, three of the primer and probe combinations for detecting mutation P681H, and three of the primers and probes for detecting mutation HV69-70del.
- the composition simultaneously comprises one of the primer and probe combinations for detecting mutation N501Y, two of the primer and probe combinations for detecting mutation P681H, and two of the primers and probes for detecting mutation HV69-70del.
- the composition simultaneously comprises one of the primer and probe combinations for detecting mutation N501Y, two of the primer and probe combinations for detecting mutation P681H, and three of the primers and probes for detecting mutation HV69-70del.
- the composition simultaneously comprises two of the primer and probe combinations for detecting mutation N501Y, two of the primer and probe combinations for detecting mutation P681H, and three of the primers and probes for detecting mutation HV69-70del.
- the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-3, the primer and probe shown by SEQ ID NO: 10-12, and the primer and probe shown by SEQ ID NO: 19-21.
- the composition simultaneously comprises: a primer and a probe shown by SEQ ID NO: 4-6, a primer and a probe shown by SEQ ID NO: 13-15, and a primer and a probe shown by SEQ ID NO: 22-24.
- the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 7-9, the primer and probe shown by SEQ ID NO: 16-18, and the primer and probe shown by SEQ ID NO: 25-27.
- the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-6, the primer and probe shown by SEQ ID NO: 10-15, and the primer and probe shown by SEQ ID NO: 19-24.
- the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-9, the primer and probe shown by SEQ ID NO: 10-18, and the primer and probe shown by SEQ ID NO: 19-27.
- the present invention provides a composition which can be used to detect major mutation sites of SARS-CoV-2 variants and identify the mutant types by combining detection results for different sites, the composition simultaneously comprising:
- composition may further comprise:
- primer and probe combination for detecting mutation K417N is a primer and a probe shown by SEQ ID NO: 28-30;
- primer and probe combination for detecting mutation E484K is a primer and a probe shown by SEQ ID NO: 31-33.
- the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-3, the primer and probe shown by SEQ ID NO: 10-12, the primer and probe shown by SEQ ID NO: 19-21, the primer and probe shown by SEQ ID NO: 28-30, and the primer and probe shown by SEQ ID NO: 31-33.
- the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 4-6, the primer and probe shown by SEQ ID NO: 13-15, the the primer and probe shown by SEQ ID NO: 22-24, the primer and probe shown by SEQ ID NO: 28-30, and the primer and probe shown by SEQ ID NO: 31-33.
- the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 7-9, the primer and probe shown by SEQ ID NO: 16-18, the the primer and probe shown by SEQ ID NO: 25-27, the primer and probe shown by SEQ ID NO: 28-30, and the primer and probe shown by SEQ ID NO: 31-33.
- the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-3, the primer and probe shown by SEQ ID NO: 13-15, the primer and probe shown by SEQ ID NO: 22-24, the primer and probe shown by SEQ ID NO: 28-30, and the primer and probe shown by SEQ ID NO: 31-33.
- the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 7-9, the primer and probe shown by SEQ ID NO: 13-15, the primer and probe shown by SEQ ID NO: 25-27, the primer and probe shown by SEQ ID NO: 28-30, and the primer and probe shown by SEQ ID NO: 31-33.
- the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-6, the primer and probe shown by SEQ ID NO: 10-15, the primer and probe shown by SEQ ID NO: 19-24, the primer and probe shown by SEQ ID NO: 28-30, and the primer and probe shown by SEQ ID NO: 31-33.
- the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-9, the primer and probe shown by SEQ ID NO: 10-18, the primer and probe shown by SEQ ID NO: 19-27, the primer and probe shown by SEQ ID NO: 28-30, and the primer and probe shown by SEQ ID NO: 31-33.
- composition which can be used to detect major mutation sites of SARS-CoV-2 variants, the composition simultaneously comprising:
- mutant K417N forward primer as shown by SEQ ID NO: 28
- mutant K417N reverse primer as shown by SEQ ID NO: 29
- mutant K417N probe as shown by SEQ ID NO: 30;
- mutant P681H forward primer as shown by SEQ ID NO: 16
- mutant P681H reverse primer as shown by SEQ ID NO: 17
- mutant P681H probe as shown by SEQ ID NO: 18.
- the present invention further provides a composition which can be used to detect major mutation sites of SARS-CoV-2 variants and identify the mutant types by combining detection results for different sites, the composition simultaneously comprising:
- mutant L452R forward primer as shown by SEQ ID NO: 40
- mutant L452R reverse primer as shown by SEQ ID NO: 41
- mutant L452R probe as shown by SEQ ID NO: 42
- mutant P681R forward primer as shown by SEQ ID NO: 46
- mutant P681R reverse primer as shown by SEQ ID NO: 47
- mutant P681R probe as shown by SEQ ID NO: 48
- mutant L452Q forward primer as shown by SEQ ID NO: 52
- mutant L452QR reverse primer as shown by SEQ ID NO: 53
- mutant L452Q probe as shown by SEQ ID NO: 54
- mutant F490S forward primer as shown by SEQ ID NO: 58
- mutant F490S reverse primer as shown by SEQ ID NO: 59
- mutant F490S probe as shown by SEQ ID NO: 60
- mutant D950N forward primer as shown by SEQ ID NO: 67
- mutant D950N reverse primer as shown by SEQ ID NO: 68
- mutant D950N probe as shown by SEQ ID NO: 69.
- first nucleic acid composition may be included; only the second nucleic acid composition may be included; only the third nucleic acid composition may be included; only the fourth nucleic acid composition may be included; and only the fifth nucleic acid composition may be included.
- the mutant L452R forward primer as shown by SEQ ID NO: 40 the mutant L452R reverse primer as shown by SEQ ID NO: 41 and the mutant L452R probe as shown by SEQ ID NO: 42
- the mutant P681R forward primer as shown by SEQ ID NO: 46 the mutant P681R reverse primer as shown by SEQ ID NO: 47 and the mutant P681R probe as shown by SEQ ID NO: 48
- the mutant L452Q forward primer as shown by SEQ ID NO: 52 the mutant L452Q reverse primer as shown by SEQ ID NO: 53 and the mutant L452Q probe as shown by SEQ ID NO: 54
- the mutant F490S forward primer as shown by SEQ ID NO: 58 the mutant F490S reverse primer as shown by SEQ ID NO: 59
- the mutant F490S probe as shown by SEQ ID NO: 60 the mutant T487K forward primer as shown by SEQ ID NO:
- composition in the present invention By using the composition in the present invention, major mutations of novel coronavirus variants can be identified, and the variant type can be identified by a combination of detection results for different sites, so that different variant types can be treated differently, thereby leading to more effective treatment and prevention.
- the composition of the present invention in combination with a fluorescent probe method, can be used to detect multiple targets simultaneously, and has the advantages of a low cost, a high throughput, a simple operation and a short usage time.
- the description “different from each other and do not interfere with each other” means that the fluorescent groups in each probe in the composition are different, and would not affect detection of each other, that is, detection can be performed by using different channels.
- FAM, HEX, ROX and CY5 can be used, the absorbance values of these groups are not close and can allow selections of different channels, without interfering with each other.
- composition includes: an internal standard forward primer, an internal standard reverse primer and an internal standard probe for monitoring.
- the internal standard comprises a human genome internal standard.
- the composition further comprises: a human genome internal standard forward primer as shown by SEQ ID NO: 34, a human genome internal standard reverse primer as shown by SEQ ID NO: 35, and a human genome internal standard probe as shown by SEQ ID NO: 36.
- the internal standard includes a novel coronavirus internal standard.
- the composition further comprises: a novel coronavirus internal standard forward primer as shown by SEQ ID NO: 37, a novel coronavirus internal standard reverse primer as shown by SEQ ID NO: 38, and a novel coronavirus internal standard probe as shown by SEQ ID NO: 39.
- the composition further comprises: a novel coronavirus internal standard forward primer as shown by SEQ ID NO: 70, a novel coronavirus internal standard reverse primer as shown by SEQ ID NO: 71, and a novel coronavirus internal standard probe as shown by SEQ ID NO: 72.
- the internal standard includes a human genome internal standard and a novel coronavirus internal standard.
- the composition further comprises: a human genome internal standard forward primer as shown by SEQ ID NO: 34, a human genome internal standard reverse primer as shown by SEQ ID NO: 35, and a human genome internal standard probe as shown by SEQ ID NO: 36; and a novel coronavirus internal standard forward primer as shown by SEQ ID NO: 37, a novel coronavirus internal standard reverse primer as shown by SEQ ID NO: 38, and a novel coronavirus internal standard probe as shown by SEQ ID NO: 39.
- the fluorescent groups of the internal standard probe and other component probes are different from each other and do not interfere with each other.
- the fluorescent reporter group may be selected from FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3 and JOE, but is not limited thereto.
- the 3' end of the probe also has a quenching group, such as BHQ1 or BHQ2.
- the 3' end of the probe is BHQ1.
- the amount of the primer used in the composition is 0.1-0.4 ⁇ M; and the amount of probe used in the composition is 0.1-0.3 ⁇ M.
- the amount of the primer used in the composition is 0.1-0.3 ⁇ M; and the amount of probe used in the composition is 0.15-0.25 ⁇ M.
- the amount of the primer used in the composition is 0.2-0.4 ⁇ M; and the amount of probe used in the composition is 0.1-0.2 ⁇ M.
- the components of the composition of the present invention are each present in separate packages.
- composition of the present invention are present in a mixed form.
- the present invention provides a use of the above described composition of the present invention in preparing a kit for detecting major mutation sites of SARS-CoV-2 variants.
- the present invention provides a kit for detecting major mutation sites of SARS-CoV-2 variants, the kit comprising the composition of the present invention as described above.
- kit further comprises a negative quality control and a positive quality control.
- the negative quality control is at least one of DEPC H 2 O, normal saline, and an internal reference gene pseudovirus.
- the positive quality control is at least one of an ORF 1ab target gene of the novel coronavirus, a N target gene of the novel coronavirus, each mutation site of the novel coronavirus, a target fragment plasmid of an internal reference gene, a fragment RNA, and a pseudovirus.
- kit further includes: a nucleic acid release system and a nucleic acid amplification system.
- the kit further comprises at least one of a dNTP, a PCR buffer, and Mg 2+ .
- the kit further includes: at least one of a nucleic acid release agent, a nucleic acid extraction reagent, a reverse transcriptase, a uracil glycosylase, and DNA polymerase.
- the kit also includes at least one of a nucleic acid release reagent, a nucleic acid extraction reagent, a dNTP, a reverse transcriptase, a uracil glycosylase, a DNA polymerase, a PCR buffer and Mg 2+ .
- the concentration of the reverse transcriptase is 5U/reaction-15U/reaction, for example, the reverse transcriptase can be murine leukemia reverse transcriptase (MMLV) or a Tth enzyme; and the concentration of the DNA polymerase is 3U/reaction-15U /reaction, for example, the DNA polymerase can be a Taq enzyme.
- the reverse transcriptase can be murine leukemia reverse transcriptase (MMLV) or a Tth enzyme
- the concentration of the DNA polymerase is 3U/reaction-15U /reaction, for example, the DNA polymerase can be a Taq enzyme.
- the kit of the present invention includes: reverse transcriptase, a Taq enzyme, a uracil glycosylase, Mg 2+ , Mn 2+ , RNasin, a dNTP, a primer, a probe and a PCR buffer.
- a conventional PCR buffer is composed of buffer systems such as Tris-HCl, MgCl2, KCl, and Triton X-100. Generally, the total volume of a single PCR reaction tube is 20-100 ⁇ l.
- the kit of the present invention is compatible with a digital PCR amplification system, that is, it can be directly used for amplification by a digital PCR machine.
- a method for detecting major mutation sites of SARS-CoV-2 variants comprising the steps of:
- step 2) performing fluorescence quantitative PCR analysis on the nucleic acids obtained in step 1) by using the above-mentioned composition of the present invention or the above-mentioned kit of the present invention;
- the sample for detection can be a throat swab, an oropharyngeal swab, a nasopharyngeal swab, sputum, a bronchoalveolar lavage fluid, blood, etc., but is not limited thereto.
- reaction conditions for the fluorescence quantitative PCR are as follows:
- the reaction conditions for the fluorescence quantitative PCR are as follows: reverse transcription at a temperature of 50°C for 10 min for 1 cycle; cDNA pre-denaturation at a temperature of 95°C for 1-10 min for 1 cycle; denaturation at a temperature of 95°C for 10 s; and annealing at a temperature of 60°C for 20 s for 45 cycles, and fluorescence is collected.
- a method for detecting major mutation sites of SARS-CoV-2 variants for non-diagnostic purposes comprising the steps of:
- step 2) performing fluorescence quantitative PCR analysis on the nucleic acids obtained in step 1) by using the above-mentioned composition of the present invention or the above-mentioned kit of the present invention;
- reaction conditions for the fluorescence quantitative PCR are as follows:
- the reaction conditions for the fluorescence quantitative PCR are as follows: reverse transcription at a temperature of 50°C for 10 min for 1 cycle; cDNA pre-denaturation at a temperature of 95°C for 1-10 min for 1 cycle; denaturation at a temperature of 95°C for 10 s; and annealing at a temperature of 60°C for 20 s for 45 cycles, and fluorescence is collected.
- non-diagnostic purpose means that it is not intended to obtain information on whether an individual is infected with a SARS-CoV-2 variant and is suffering from pneumonia.
- the method could detect presence of SARS-CoV-2 variants in test cultures in experiments for research purposes.
- the biological sample is a body fluid.
- the body fluid can be a fluid isolated from anywhere in a subject's body (for example, peripheral sites) , including, but not limited to, for example, blood, blood plasma, blood serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural effusion, nipple aspirate fluid, lymphatic fluid, fluids of the respiratory, intestinal and urogenital tracts, tears, saliva, breast milk, fluids from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluids, ascites, tumor cyst fluid, amniotic fluid, and combinations thereof.
- the body fluid may be urine, blood serum, or cerebrospinal fluid.
- Clinical samples should be respiratory tract samples (especially lower respiratory tract samples) collected during the early stages of the onset of a case, the acute-phase blood serum within 7 days of the onset, and the convalescent blood serum at 3rd to 4th week after the onset of the disease.
- Figures 1-4 are detection results of detected samples (N501Y and HV69-70del, P681H, E484K, and K417N, respectively) of compositions in Table 3 of the present invention.
- Figures 5-11 are detection results of the detected samples (N gene, E484K, P681R, L452R and E484Q, T478K and D950N, L452Q and T76I, and F490S and Del247-253) of compositions in Table 4 of the present invention.
- Figure 12 is a corresponding amplification curve when amplifying a positive sample with mutations by a primer and probe combination in Table 1 of the present invention.
- Figure 13 is a corresponding amplification curve when amplifying a positive sample with mutations by a primer and probe combination in Table 2 of the present invention.
- Figure 14 is a corresponding amplification curve when amplifying a negative sample without mutation by a primer and probe combination in Table 1 of the present invention.
- Figure 15 is a corresponding amplification curve when amplifying a negative sample without mutation by a primer and probe combination in Table 2 of the present invention.
- Figures 16-22 are detection results for detecting samples with different concentrations (i.e. sensitivity) by compositions in Tables 1-4 of the present invention.
- Figures 23-24 are specificity detection results of compositions in Tables 3-4 of the present invention.
- Figures 25-26 are accuracy detection results of compositions (E484K and K417N, respectively) in Table 3 of the present invention.
- Figures 27-28 are accuracy detection results of compositions (L452R, E484Q) of Table 4 of the present invention.
- Figures 29-32 are detection results of comparative compositions (E484K, K417N, P681H, and HV69-70del, respectively) in Table 3 of the present invention.
- Figures 33-35 are detection results of detected samples of comparative compositions (L452R, E484Q, and E484K) in Table 4 of the present invention.
- useful primers and probes have nucleotide sequences having greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with the primers or probes provided in Tables 1-4. Modifications to such primers and probes are also expected and can be prepared according to standard techniques.
- %identity refers to two or more sequences or sub-sequences which are identical or have a specified percentage of identical amino acid residues or nucleotides when comparison and alignment are performed for homology, as measured using one of the following sequence comparison algorithms or by visual inspection. For example, %identity is measured with respect to the entire length of the coding region of the sequences being compared.
- sequence comparison typically one sequence is used as a reference sequence, to which a test sequence is compared.
- test and reference sequences are input into a computer, and if necessary, sub-sequence coordinates are designated, and sequence algorithm program parameters are designated. Then, the sequence comparison algorithm is used to calculate the percent sequence identity of the test sequence relative to the reference sequence according to the specified program parameters.
- a search algorithm such as BLAST and PSI -BLAST (Altschul et al., 1990, Mol Biol 215: 3, 403-410; Altschul et al., 1997, Nucleic Acids Res25: 17, 3389-402) can be used to determine percent identity.
- primers and probes can be modified by known methods. Modified versions of these primer and/or probe sequences may include, by way of non-limiting examples, addition of one or more nucleotides to the 5' end, addition of one or more nucleotides to the 3' end, addition of one or more nucleotides to the 5' and 3' ends, addition of tails, shortening of the sequence, lengthening of the sequence, shifting of the sequence forward or backward by a few bases, or any combination thereof.
- Base modifications include, for example, 3'P, 5'P, 5-nitroindole, 2-aminopurine, 8-amino-2'-deoxyadenosine, C-5 propynyl-deoxycytidine, C-5 propynyl-deoxyuridine, 2-amino-2'-deoxyadenosine-5'-triphosphate, 2, 6-diaminopurine (2-amino-dA) , reverse dT, reverse dideoxy-T, hydroxymethyl dC, iso-dC, 5-methyl dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNA's) , and include at least one mismatched base at one of the bases, or a replacement of at least one of the bases with an RNA base, so as to achieve, for example, increasing nucleic acid interaction at the 3' end of the mutant-specific primer to increase Tm.
- LNA's locked nucleic acids
- the fluorescent group of NY-P is FAM; the fluorescent group of HV-P is HEX; the fluorescent group of PH-P is CY5; and the fluorescent gene of N-P is ROX.
- the fluorescent group of NY-P is FAM; the fluorescent group of HV-P is HEX; the fluorescent group of PH-P is CY5; and the fluorescent gene of N-P is ROX.
- the fluorophore of N501Y-P and E484K-P is FAM; the fluorophore of HV69-70Del-P and K417N-P is HEX; the fluorophore of P681H-P is CY5, the internal standard N-P probe of the novel coronavirus is labeled as a ROX channel, and the human gene internal standard IC-P probe is labeled as a CY5 channel.
- L452R-P, P681R-P, L452Q-P, F490S-P and T478K-P probes are labeled as FAM channels
- E484Q-P, E484K-P, T76I-P, Del247-253-P and D950N-P probes are labeled as HEX channels
- the internal standard N-P probe of novel coronavirus is labeled as a ROX channel
- the human gene internal standard IC-P probe is labeled as a CY5 channel.
- a detection samples in the present invention was a throat swab, sputum, a bronchoalveolar lavage fluid, or blood.
- Viral nucleic acids were extracted by a magnetic bead method (a kit from Sansure Biotech INC. ) , and the following operations were performed in a sample treatment chamber:
- RNA extraction solution 2-mix 100 ⁇ L was added to each tube (sucked up after thorough mixing) , and the tube was shaken for 10 seconds for thorough mixing, and left to stand for 10 minutes at room temperature.
- RNA extraction solution 3 2.5 600 ⁇ L of an RNA extraction solution 3 and 200 ⁇ L of an RNA extraction solution 4 were added to each tube, and the tubes were shaken for 5 seconds for thorough mixing, and subjected to instant centrifugation, and the centrifuged tubes were placed on the separator again.
- PCR-mix 2.7 50 ⁇ L of PCR-mix was added to each tube, and a pipette was used to suck up the PCR-mix to elute the brown residue adhered to the wall of the centrifuged tube. The operation was repeated several times to elute the residue as completely as possible, and then all the eluted brown mixture was transferred to a 0.2mL PCR reaction tube, which was then covered with a cap and transferred to an amplification detection zone.
- the real-time fluorescent PCR reaction system was formulated as follows:
- the PCR amplification procedure was set up as follows:
- the target detection signals were FAM, HEX (or VIC) and ROX and the internal reference detection signal was CY5/ROX;
- Baseline setting the baseline was generally set to 3-15 cycles, depending on the actual situations.
- the adjustment principle was: selecting a zone with a relatively sTable fluorescence signal before exponential amplification, to enable a starting point (Start) to avoid signal fluctuation during an initial stage of fluorescence acquisition, and to allow an end point (End) with cycles 1-2 less than Ct of the earliest exponentially amplified sample.
- Threshold setting the setting principle was to make the threshold line just exceed the highest point of the normal negative control.
- compositions in Table 3 of the present invention were subjected to fluorescence quantitative PCR to detect several major mutation sites for novel coronavirus nucleic acids, including mutation sites such as E484K, K417N, P681H, N501Y and HV69-70del, one of the samples was found to have typical mutations such as N501Y, HV69-70del and P681H (figures 1-2) , and one sample was also found to have E484 and K417N mutations (figures 3-4) . It can be seen from the figures that corresponding targets can be detected by the composition of the present invention according to determination rule 1, proving that the composition of the present invention can be used to detect novel coronavirus variants.
- samples positive samples: artificially synthesized nucleic acid sequences containing N501Y, HV69-70del and P681H mutations; and negative samples: nucleic acids with no N501Y, HV69 -70del and P681H mutations extracted from inactivated novel coronavirus, and concentration calibration was performed by novel coronavirus nucleic acid detection kits) were detected, and the results are shown in figures 12-15. It can be seen from the figures that corresponding targets can be detected, proving that the composition of the present invention can be used to detect novel coronavirus variants.
- compositions in Tables 1-4 of the present invention By using the compositions in Tables 1-4 of the present invention and according to the method described in example 2, detection was performed at 5 concentrations, 200,000, 20,000, 20,000, 20,000, 200, and 20 copies/ml of each target pseudovirus. Multiple PCR tests were performed by a Hongshi fluorescent quantitative PCR instrument, and the results are shown in figures 16-17 (compositions in Tables 1 and 2) , 18-20 (compositions in Table 3) , and 21-22 (compositions in Table 4) . It can be seen from the figures that corresponding targets can still be detected at a concentration as low as 200 copies/ml, proving that the sensitivity of the composition of the present invention is 200 copies/ml.
- compositions of Tables 1-4 of the present invention were performed by a Hongshi fluorescent quantitative PCR instrument for pathogens which have homologous nucleic acid sequences and are prone to cause the same or similar clinical symptoms (such as coronaviruses (NL63, HKU1, 229E and 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.
- coronaviruses NL63, HKU1, 229E and OC43
- dimers Due to the principle of complementary base pairing, dimers can be formed by primers and/or probes, but this probability is low and can be ruled out at the beginning of the design. However, when multiple pathogens are jointly detected, there are numerous primers and probes, and dimers are prone to form between a primer and a primer, a probe and a probe, or a primer and a probe. In order to ensure the conservation of the design (conservation is critical to the accuracy of detection) and take mutual interference between primers and probes into consideration, the primers and probes need to be carefully designed.
- the inventor also designed other primers and probes (sequences not shown) to form different detection systems 1-4 (comparative examples to compositions in Table 3) , and 5-7 (comparative examples to compositions in Table 4) , which were also used to detect novel coronavirus mutations.
- Specific detection results are shown in figures 29-32 (comparative examples to compositions in Table 3) and 33-35 (comparative examples to compositions in Table 4) . It can be seen from the figures that the detection effects are poor.
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Abstract
The present invention relates to the field of molecular biology detection, specifically to detection of SARS-CoV-2, and more specifically to detection of major mutation sites of SARS-CoV-2 variants. Further provided are a kit comprising a composition, a use of the composition, and a method for detecting and genotyping of SARS-CoV-2 variants. By using the composition in the present invention, major mutations of the novel coronavirus variants can be identified, and preliminary screening and confirmation of variant types are performed by site-combination detection, so that different mutation sites can be treated differently, thereby leading to more effective treatment and prevention. The composition of the present invention, in combination with a fluorescent probe method, can be used to detect multiple targets simultaneously, with advantages of a low cost, a high throughput, a simple operation and a short usage time.
Description
- CROSS-REFERENCE TO RELATED APPLICATIONS
- The present application claims priority to: the application No. 202111500433. X, filed on December 09, 2021, entitled “Composition, Kit, Method and Use thereof for Detecting SARS-CoV-2 Mutation Sites” , the application No. 202110592489.6, filed on May 28, 2021, entitled “Combination Product, Kit, Use and Method for Detecting Mutant SARS-CoV-2 Viruses” , the application No. 202111503790.1, filed on December 10, 2021, entitled “Composition, Kit, Method and Use thereof for Detecting SARS-CoV-2 Mutation Sites” , and the application No. 202111667275.7, filed on December 31, 2021, and entitled “Combination Product, Kit, Use and Method for Detecting Mutant SARS-CoV-2 Viruses” , the above Chinese patent applications being incorporated by reference into the present application in their entireties.
- The present invention relates to the field of molecular biology detection, specifically to detection of SARS-CoV-2, and more specifically to detection of major mutation sites of SARS-CoV-2 variants.
- As an RNA virus, one of the significant characteristics of the novel coronavirus is easy mutation, from variant alpha (Alpha B. 1.1.7) to beta (Beta B. 1.351) , again to gamma (Gamma P. 1) and delta (Delta B. 1.617.2) , after each mutation, the virus attains stronger virus transmissibility. Currently, the Delta variant found in India is becoming the most prevalent variant of the novel coronavirus in the world due to its significantly enhanced transmission ability. The new variant evades the line of defense of many countries, such as east Asian countries including China, and southeast Asian countries, and after breaking the line of defense of public health prevention and control, the variant becomes a challenge to the vaccine defensive line.
- Based on global sequencing analysis reports of SARS-CoV-2 genomes, mutations thereof mainly occur at 5 genes, i.e., S gene, N gene, ORF8 gene, ORF3a gene and ORF1ab gene, wherein the mutation on S gene has a greatest effect on the transmission and pathogenicity of the virus. At present, the most prevalent variants mainly include B. 1.1.7, B. 1.351, B. 1.617.1, B.1.617.2, B. 1.617.3, B. 1.525, P. 1, P. 2, C. 37 and the like. Major mutation sites are E484Q, E484K, L452R, P681R, T487K, D950N, F490S, Del247-253, T76I, L452Q, and the like, and these sites may lead to increased immune escape or infectivity. In particular, after emergence of Delta and Lambda variants, said variants are spreading rapidly and are gradually superseding other types novel coronavirus. A huge challenge is generated for epidemic prevention and control, and development of vaccines is also urgent. Some of the mutations may be resistant to antibodies induced by current SARS-CoV-2 vaccines, and affect the efficacy of detection reagents and vaccines. Therefore, identifying mutation sites of the virus is of great significance to epidemiological analysis and clinical diagnosis of the virus, and treatment related thereto.
- A reagent is required in the art, which can be used to accurately identify different mutations of the novel coronavirus, so that targeted epidemic prevention and treatment measures can be taken to make the response more efficient. Moreover, the detection time must be short and the sensitivity must be high.
- SUMMARY
- In a first aspect, the present invention provides a composition which can be used to detect major mutation sites of SARS-CoV-2 variants and identify the mutant types by combining detection results for different sites, the composition simultaneously comprising:
- a primer and probe combination for detecting mutation N501Y, a primer and probe combination for detecting mutation P681H, and a primer and probe combination for detecting mutation HV69-70del;
- wherein the primer and probe combination for detecting mutation N501Y is selected from one or more of the following groups:
- a primer and a probe shown by SEQ ID NO: 1-3, a primer and a probe shown by SEQ ID NO: 4-6, and a primer and a probe shown by SEQ ID NO: 7-9;
- wherein the primer and probe combination for detecting mutation P681H is selected from one or more of the following groups:
- a primer and a probe shown by SEQ ID NO: 10-12, a primer and a probe shown by SEQ ID NO: 13-15, and a primer and a probe shown by SEQ ID NO: 16-18;
- wherein, the primer and probe combination for detecting mutation HV69-70del is selected from one or more of the following groups:
- a primer and a probe shown by SEQ ID NO: 19-21, a primer and a probe shown by SEQ ID NO: 22-24, and a primer and a probe shown by SEQ ID NO: 25-27.
- Further, in some embodiments, the composition of the present invention may simultaneously include one or more pairs of the primers and probes described above. In the present invention, “pair” refers to mutually matched upstream and reverse primers and probes for detecting mutations.
- In some specific embodiments, the composition can include one, two, or three primer and probe combinations for detecting mutation N501Y.
- In some specific embodiments, the composition can include one, two, or three primer and probe combinations for detecting mutation P681H.
- In some specific embodiments, the composition can include one, two, or three primer and probe combinations for detecting mutation HV69-70del.
- For example, in some specific embodiments, the composition simultaneously comprises one of the primer and probe combinations for detecting mutation N501Y, one of the primer and probe combinations for detecting mutation P681H, and one of the primers and probes for detecting mutation HV69-70del.
- For example, in some specific embodiments, the composition simultaneously comprises two of the primer and probe combinations for detecting mutation N501Y, two of the primer and probe combinations for detecting mutation P681H, and two of primers and probes for detecting mutation HV69-70del.
- For example, in some specific embodiments, the composition simultaneously comprises three of the primer and probe combinations for detecting mutation N501Y, three of the primer and probe combinations for detecting mutation P681H, and three of the primers and probes for detecting mutation HV69-70del.
- For example, in some specific embodiments, the composition simultaneously comprises one of the primer and probe combinations for detecting mutation N501Y, two of the primer and probe combinations for detecting mutation P681H, and two of the primers and probes for detecting mutation HV69-70del.
- For example, in some specific embodiments, the composition simultaneously comprises one of the primer and probe combinations for detecting mutation N501Y, two of the primer and probe combinations for detecting mutation P681H, and three of the primers and probes for detecting mutation HV69-70del.
- For example, in some specific embodiments, the composition simultaneously comprises two of the primer and probe combinations for detecting mutation N501Y, two of the primer and probe combinations for detecting mutation P681H, and three of the primers and probes for detecting mutation HV69-70del.
- For example, in a specific embodiment, the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-3, the primer and probe shown by SEQ ID NO: 10-12, and the primer and probe shown by SEQ ID NO: 19-21.
- For example, in a specific embodiment, the composition simultaneously comprises: a primer and a probe shown by SEQ ID NO: 4-6, a primer and a probe shown by SEQ ID NO: 13-15, and a primer and a probe shown by SEQ ID NO: 22-24.
- For example, in a specific embodiment, the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 7-9, the primer and probe shown by SEQ ID NO: 16-18, and the primer and probe shown by SEQ ID NO: 25-27.
- For example, in a specific embodiment, the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-6, the primer and probe shown by SEQ ID NO: 10-15, and the primer and probe shown by SEQ ID NO: 19-24.
- For example, in a specific embodiment, the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-9, the primer and probe shown by SEQ ID NO: 10-18, and the primer and probe shown by SEQ ID NO: 19-27.
- In a specific embodiment, the present invention provides a composition which can be used to detect major mutation sites of SARS-CoV-2 variants and identify the mutant types by combining detection results for different sites, the composition simultaneously comprising:
- a) and/or b) :
- a) the primer and probe shown by SEQ ID NO: 1-3, the primer and probe shown by SEQ ID NO: 10-12, and the primer and probe shown by SEQ ID NO: 19-21; and
- b) the primer and probe shown by SEQ ID NO: 4-6, the primer and probe shown by SEQ ID NO: 13-15, and the primer and probe shown by SEQ ID NO: 22-24.
- Further, the composition may further comprise:
- a primer and probe combination for detecting mutation K417N and a primer and probe combination for detecting mutation E484K;
- wherein the primer and probe combination for detecting mutation K417N is a primer and a probe shown by SEQ ID NO: 28-30; and
- wherein the primer and probe combination for detecting mutation E484K is a primer and a probe shown by SEQ ID NO: 31-33.
- For example, in a specific embodiment, the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-3, the primer and probe shown by SEQ ID NO: 10-12, the primer and probe shown by SEQ ID NO: 19-21, the primer and probe shown by SEQ ID NO: 28-30, and the primer and probe shown by SEQ ID NO: 31-33.
- For example, in a specific embodiment, the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 4-6, the primer and probe shown by SEQ ID NO: 13-15, the the primer and probe shown by SEQ ID NO: 22-24, the primer and probe shown by SEQ ID NO: 28-30, and the the primer and probe shown by SEQ ID NO: 31-33.
- For example, in a specific embodiment, the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 7-9, the primer and probe shown by SEQ ID NO: 16-18, the the primer and probe shown by SEQ ID NO: 25-27, the primer and probe shown by SEQ ID NO: 28-30, and the the primer and probe shown by SEQ ID NO: 31-33.
- For example, in a specific embodiment, the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-3, the primer and probe shown by SEQ ID NO: 13-15, the primer and probe shown by SEQ ID NO: 22-24, the primer and probe shown by SEQ ID NO: 28-30, and the primer and probe shown by SEQ ID NO: 31-33.
- For example, in a specific embodiment, the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 7-9, the primer and probe shown by SEQ ID NO: 13-15, the primer and probe shown by SEQ ID NO: 25-27, the primer and probe shown by SEQ ID NO: 28-30, and the primer and probe shown by SEQ ID NO: 31-33.
- For example, in a specific embodiment, the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-6, the primer and probe shown by SEQ ID NO: 10-15, the primer and probe shown by SEQ ID NO: 19-24, the primer and probe shown by SEQ ID NO: 28-30, and the primer and probe shown by SEQ ID NO: 31-33.
- For example, in a specific embodiment, the composition simultaneously comprises: the primer and probe shown by SEQ ID NO: 1-9, the primer and probe shown by SEQ ID NO: 10-18, the primer and probe shown by SEQ ID NO: 19-27, the primer and probe shown by SEQ ID NO: 28-30, and the primer and probe shown by SEQ ID NO: 31-33.
- In a specific embodiment, a composition is provided, which can be used to detect major mutation sites of SARS-CoV-2 variants, the composition simultaneously comprising:
- a first nucleic acid composition:
- a mutant N501Y forward primer as shown by SEQ ID NO: 7, a mutant N501Y reverse primer as shown by SEQ ID NO: 8, and a mutant N501Y probe as shown by SEQ ID NO: 9; and
- a mutant HV69-70del forward primer shown by SEQ ID NO: 25, a mutant HV69-70del reverse primer shown by SEQ ID NO: 26, and a mutant HV69-70del probe shown by SEQ ID NO: 27; and
- a second nucleic acid composition:
- a mutant K417N forward primer as shown by SEQ ID NO: 28, a mutant K417N reverse primer as shown by SEQ ID NO: 29, and a mutant K417N probe as shown by SEQ ID NO: 30;
- a mutant E484K forward primer as shown by SEQ ID NO: 31, a mutant E484K reverse primer as shown by SEQ ID NO: 32, and a mutant E484K probe as shown by SEQ ID NO: 33; and
- a mutant P681H forward primer as shown by SEQ ID NO: 16, a mutant P681H reverse primer as shown by SEQ ID NO: 17, and a mutant P681H probe as shown by SEQ ID NO: 18.
- In addition, the present invention further provides a composition which can be used to detect major mutation sites of SARS-CoV-2 variants and identify the mutant types by combining detection results for different sites, the composition simultaneously comprising:
- a first nucleic acid composition:
- a mutant L452R forward primer as shown by SEQ ID NO: 40, a mutant L452R reverse primer as shown by SEQ ID NO: 41, and a mutant L452R probe as shown by SEQ ID NO: 42; and
- a mutant E484Q forward primer as shown by SEQ ID NO: 43, a mutant E484Q reverse primer as shown by SEQ ID NO: 44, and a mutant E484Q probe as shown by SEQ ID NO: 45;
- a second nucleic acid composition:
- a mutant P681R forward primer as shown by SEQ ID NO: 46, a mutant P681R reverse primer as shown by SEQ ID NO: 47, and a mutant P681R probe as shown by SEQ ID NO: 48; and
- a mutant E484K forward primer as shown by SEQ ID NO: 49, a mutant E484K reverse primer as shown by SEQ ID NO: 50, and a mutant E484K probe as shown by SEQ ID NO: 51;
- a third nucleic acid composition:
- a mutant L452Q forward primer as shown by SEQ ID NO: 52, a mutant L452QR reverse primer as shown by SEQ ID NO: 53, and a mutant L452Q probe as shown by SEQ ID NO: 54; and
- a mutant T76I forward primer as shown by SEQ ID NO: 55, a mutant T76I reverse primer as shown by SEQ ID NO: 56, and a mutant T76I probe as shown by SEQ ID NO: 57;
- a fourth nucleic acid composition:
- a mutant F490S forward primer as shown by SEQ ID NO: 58, a mutant F490S reverse primer as shown by SEQ ID NO: 59, and a mutant F490S probe as shown by SEQ ID NO: 60; and
- a mutant Del247-253 forward primer as shown by SEQ ID NO: 61, a mutant Del247-253 reverse primer as shown by SEQ ID NO: 62, and a mutant Del247-253 probe as shown by SEQ ID NO: 63; and
- a fifth nucleic acid composition:
- a mutant T487K forward primer as shown by SEQ ID NO: 64, a mutant T487K reverse primer as shown by SEQ ID NO: 65, and a mutant T487K probe as shown by SEQ ID NO: 66; and
- a mutant D950N forward primer as shown by SEQ ID NO: 67, a mutant D950N reverse primer as shown by SEQ ID NO: 68, and a mutant D950N probe as shown by SEQ ID NO: 69.
- For example, only the first nucleic acid composition may be included; only the second nucleic acid composition may be included; only the third nucleic acid composition may be included; only the fourth nucleic acid composition may be included; and only the fifth nucleic acid composition may be included.
- For example, only some primer and probe pairs of different nucleic acid compositions are included, such as, the mutant L452R forward primer as shown by SEQ ID NO: 40, the mutant L452R reverse primer as shown by SEQ ID NO: 41 and the mutant L452R probe as shown by SEQ ID NO: 42, the mutant P681R forward primer as shown by SEQ ID NO: 46, the mutant P681R reverse primer as shown by SEQ ID NO: 47 and the mutant P681R probe as shown by SEQ ID NO: 48, the mutant L452Q forward primer as shown by SEQ ID NO: 52, the mutant L452Q reverse primer as shown by SEQ ID NO: 53 and the mutant L452Q probe as shown by SEQ ID NO: 54, and the mutant F490S forward primer as shown by SEQ ID NO: 58, the mutant F490S reverse primer as shown by SEQ ID NO: 59, and the mutant F490S probe as shown by SEQ ID NO: 60, the mutant T487K forward primer as shown by SEQ ID NO: 64, the mutant T487K reverse primer as shown by SEQ ID NO: 65, and the mutant T487K probe as shown by SEQ ID NO: 66.
- By using the composition in the present invention, major mutations of novel coronavirus variants can be identified, and the variant type can be identified by a combination of detection results for different sites, so that different variant types can be treated differently, thereby leading to more effective treatment and prevention. The composition of the present invention, in combination with a fluorescent probe method, can be used to detect multiple targets simultaneously, and has the advantages of a low cost, a high throughput, a simple operation and a short usage time.
- Those skilled in the art could select a design where the fluorescent groups of the probes in the composition are different from each other and do not interfere with each other according to specific needs.
- In this context, the description “different from each other and do not interfere with each other” means that the fluorescent groups in each probe in the composition are different, and would not affect detection of each other, that is, detection can be performed by using different channels. For example, FAM, HEX, ROX and CY5 can be used, the absorbance values of these groups are not close and can allow selections of different channels, without interfering with each other.
- Further, the composition includes: an internal standard forward primer, an internal standard reverse primer and an internal standard probe for monitoring.
- Further, the internal standard comprises a human genome internal standard.
- In a specific embodiment, the composition further comprises: a human genome internal standard forward primer as shown by SEQ ID NO: 34, a human genome internal standard reverse primer as shown by SEQ ID NO: 35, and a human genome internal standard probe as shown by SEQ ID NO: 36.
- Further, the internal standard includes a novel coronavirus internal standard.
- In a specific embodiment, the composition further comprises: a novel coronavirus internal standard forward primer as shown by SEQ ID NO: 37, a novel coronavirus internal standard reverse primer as shown by SEQ ID NO: 38, and a novel coronavirus internal standard probe as shown by SEQ ID NO: 39.
- In a specific embodiment, the composition further comprises: a novel coronavirus internal standard forward primer as shown by SEQ ID NO: 70, a novel coronavirus internal standard reverse primer as shown by SEQ ID NO: 71, and a novel coronavirus internal standard probe as shown by SEQ ID NO: 72.
- Further, the internal standard includes a human genome internal standard and a novel coronavirus internal standard.
- In a specific embodiment, the composition further comprises: a human genome internal standard forward primer as shown by SEQ ID NO: 34, a human genome internal standard reverse primer as shown by SEQ ID NO: 35, and a human genome internal standard probe as shown by SEQ ID NO: 36; and a novel coronavirus internal standard forward primer as shown by SEQ ID NO: 37, a novel coronavirus internal standard reverse primer as shown by SEQ ID NO: 38, and a novel coronavirus internal standard probe as shown by SEQ ID NO: 39.
- Further, the fluorescent groups of the internal standard probe and other component probes 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.
- Further, the amount of the primer used in the composition is 0.1-0.4 μM; and the amount of probe used in the composition is 0.1-0.3 μM.
- Further, the amount of the primer used in the composition is 0.1-0.3 μM; and the amount of probe used in the composition is 0.15-0.25 μM.
- Further, the amount of the primer used in the composition is 0.2-0.4 μM; and the amount of probe used in the composition is 0.1-0.2 μM.
- In a specific embodiment, the components of the composition of the present invention are each present in separate packages.
- Further, the components of the composition of the present invention are present in a mixed form.
- In a second aspect, the present invention provides a use of the above described composition of the present invention in preparing a kit for detecting major mutation sites of SARS-CoV-2 variants.
- In a third aspect, the present invention provides a kit for detecting major mutation sites of SARS-CoV-2 variants, the kit comprising the composition of the present invention as described above.
- Further, the kit further comprises a negative quality control and a positive quality control.
- In a specific embodiment, the negative quality control is at least one of DEPC H 2O, normal saline, and an internal reference gene pseudovirus. The positive quality control is at least one of an ORF 1ab target gene of the novel coronavirus, a N target gene of the novel coronavirus, each mutation site of the novel coronavirus, a target fragment plasmid of an internal reference gene, a fragment RNA, and a pseudovirus.
- Further, the kit further includes: a nucleic acid release system and a nucleic acid amplification system.
- Further, the kit further comprises at least one of a dNTP, a PCR buffer, and Mg 2+.
- Further, the kit further includes: at least one of a nucleic acid release agent, a nucleic acid extraction reagent, a reverse transcriptase, a uracil glycosylase, and DNA polymerase.
- Still further, the kit also includes at least one of a nucleic acid release reagent, a nucleic acid extraction reagent, a dNTP, a reverse transcriptase, a uracil glycosylase, a DNA polymerase, a PCR buffer and Mg 2+.
- 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 a Tth enzyme; and the concentration of the DNA polymerase is 3U/reaction-15U /reaction, for example, the DNA polymerase can be a Taq enzyme.
- In a specific embodiment, the kit of the present invention includes: reverse transcriptase, a Taq enzyme, a uracil glycosylase, Mg 2+, Mn 2+, RNasin, a dNTP, a primer, a probe and a PCR buffer.
- A conventional PCR buffer is composed of buffer systems such as Tris-HCl, MgCl2, KCl, and Triton X-100. Generally, the total volume of a single PCR reaction tube is 20-100μl.
- In a specific embodiment, the kit of the present invention is compatible with a digital PCR amplification system, that is, it can be directly used for amplification by a digital PCR machine.
- In a fourth aspect, a method is provided for detecting major mutation sites of SARS-CoV-2 variants, the method comprising the steps of:
- 1) extracting or releasing nucleic acids in a sample to be tested;
- 2) performing fluorescence quantitative PCR analysis on the nucleic acids obtained in step 1) by using the above-mentioned composition of the present invention or the above-mentioned kit of the present invention; and
- 3) obtaining and analyzing the results.
- In the present invention, the sample for detection can be a throat swab, an oropharyngeal swab, a nasopharyngeal swab, sputum, a bronchoalveolar lavage fluid, blood, etc., but is not limited thereto.
- Further, the reaction conditions for the fluorescence quantitative PCR are as follows:
- reverse transcription at a temperature of 50-60℃ for 5-30 min for 1 cycle; cDNA pre-denaturation at a temperature of 95℃ for 1-10 min for 1 cycle; denaturation at a temperature of 95℃ for 5-20 s; and annealing at a temperature of 55-60℃ for 20-60 s for 40-50 cycles, and fluorescence is collected.
- In a specific embodiment, the reaction conditions for the fluorescence quantitative PCR are as follows: reverse transcription at a temperature of 50℃ for 10 min for 1 cycle; cDNA pre-denaturation at a temperature of 95℃ for 1-10 min for 1 cycle; denaturation at a temperature of 95℃ for 10 s; and annealing at a temperature of 60℃ for 20 s for 45 cycles, and fluorescence is collected.
- In a specific embodiment, a method is provided for detecting major mutation sites of SARS-CoV-2 variants for non-diagnostic purposes, the method comprising the steps of:
- 1) extracting or releasing nucleic acids in a sample to be tested;
- 2) performing fluorescence quantitative PCR analysis on the nucleic acids obtained in step 1) by using the above-mentioned composition of the present invention or the above-mentioned kit of the present invention; and
- 3) obtaining and analyzing results.
- Further, the reaction conditions for the fluorescence quantitative PCR are as follows:
- reverse transcription at a temperature of 50-60℃ for 5-30 min for 1 cycle; cDNA pre-denaturation at a temperature of 95℃ for 1-10 min for 1 cycle; denaturation at a temperature of 95℃ for 5-20 s; and annealing at a temperature of 55-60℃ for 20-60 s for 40-50 cycles, and fluorescence is collected.
- In a specific embodiment, the reaction conditions for the fluorescence quantitative PCR are as follows: reverse transcription at a temperature of 50℃ for 10 min for 1 cycle; cDNA pre-denaturation at a temperature of 95℃ for 1-10 min for 1 cycle; denaturation at a temperature of 95℃ for 10 s; and annealing at a temperature of 60℃ for 20 s for 45 cycles, and fluorescence is collected.
- As used herein, the term “non-diagnostic purpose” means that it is not intended to obtain information on whether an individual is infected with a SARS-CoV-2 variant and is suffering from pneumonia. For example, the method could detect presence of SARS-CoV-2 variants in test cultures in experiments for research purposes.
- In certain embodiments, the biological sample is a body fluid. The body fluid can be a fluid isolated from anywhere in a subject's body (for example, peripheral sites) , including, but not limited to, for example, blood, blood plasma, blood serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural effusion, nipple aspirate fluid, lymphatic fluid, fluids of the respiratory, intestinal and urogenital tracts, tears, saliva, breast milk, fluids from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluids, ascites, tumor cyst fluid, amniotic fluid, and combinations thereof. For example, the body fluid may be urine, blood serum, or cerebrospinal fluid. The samples used for detection of SARS-CoV-are preferably upper respiratory tract samples (such as throat swabs, nasal swabs, nasopharyngeal swabs, etc. ) , lower respiratory tract samples (such as respiratory tract aspirates, bronchial lavage fluids, alveolar lavage fluids, deep cough sputum, etc. ) , conjunctival swabs, stool samples, anal swabs, anticoagulation and blood serum samples, etc. Clinical samples should be respiratory tract samples (especially lower respiratory tract samples) collected during the early stages of the onset of a case, the acute-phase blood serum within 7 days of the onset, and the convalescent blood serum at 3rd to 4th week after the onset of the disease.
- The technical features of the aforementioned embodiments may be arbitrarily combined. For concise description, not all possible combinations of the technical features in the aforementioned embodiments are described. However, combinations of these technical features shall all be considered as falling within the scope described in this specification as long as the combinations of these technical features do not conflict with each other.
- The aforementioned embodiments describe only several implementation manners of the present invention. The description is specific and detailed, but cannot be therefore construed as a limitation to the scope of the patent for invention. It should be pointed out that a person of ordinary skill in the art can further make multiple variations and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present patent for invention shall be subject to the appended claims.
- Figures 1-4 are detection results of detected samples (N501Y and HV69-70del, P681H, E484K, and K417N, respectively) of compositions in Table 3 of the present invention.
- Figures 5-11 are detection results of the detected samples (N gene, E484K, P681R, L452R and E484Q, T478K and D950N, L452Q and T76I, and F490S and Del247-253) of compositions in Table 4 of the present invention.
- Figure 12 is a corresponding amplification curve when amplifying a positive sample with mutations by a primer and probe combination in Table 1 of the present invention.
- Figure 13 is a corresponding amplification curve when amplifying a positive sample with mutations by a primer and probe combination in Table 2 of the present invention.
- Figure 14 is a corresponding amplification curve when amplifying a negative sample without mutation by a primer and probe combination in Table 1 of the present invention.
- Figure 15 is a corresponding amplification curve when amplifying a negative sample without mutation by a primer and probe combination in Table 2 of the present invention.
- Figures 16-22 are detection results for detecting samples with different concentrations (i.e. sensitivity) by compositions in Tables 1-4 of the present invention.
- Figures 23-24 are specificity detection results of compositions in Tables 3-4 of the present invention.
- Figures 25-26 are accuracy detection results of compositions (E484K and K417N, respectively) in Table 3 of the present invention.
- Figures 27-28 are accuracy detection results of compositions (L452R, E484Q) of Table 4 of the present invention.
- Figures 29-32 are detection results of comparative compositions (E484K, K417N, P681H, and HV69-70del, respectively) in Table 3 of the present invention.
- Figures 33-35 are detection results of detected samples of comparative compositions (L452R, E484Q, and E484K) in Table 4 of the present invention.
- In one aspect, useful primers and probes have nucleotide sequences having greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identity with the primers or probes provided in Tables 1-4. Modifications to such primers and probes are also expected and can be prepared according to standard techniques.
- In the context of two or more nucleotide sequences or amino acid sequences, the term “%identity” refers to two or more sequences or sub-sequences which are identical or have a specified percentage of identical amino acid residues or nucleotides when comparison and alignment are performed for homology, as measured using one of the following sequence comparison algorithms or by visual inspection. For example, %identity is measured with respect to the entire length of the coding region of the sequences being compared.
- Regarding sequence comparison, typically one sequence is used as a reference sequence, to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, and if necessary, sub-sequence coordinates are designated, and sequence algorithm program parameters are designated. Then, the sequence comparison algorithm is used to calculate the percent sequence identity of the test sequence relative to the reference sequence according to the specified program parameters. A search algorithm such as BLAST and PSI -BLAST (Altschul et al., 1990, Mol Biol 215: 3, 403-410; Altschul et al., 1997, Nucleic Acids Res25: 17, 3389-402) can be used to determine percent identity.
- The primers and probes can be modified by known methods. Modified versions of these primer and/or probe sequences may include, by way of non-limiting examples, addition of one or more nucleotides to the 5' end, addition of one or more nucleotides to the 3' end, addition of one or more nucleotides to the 5' and 3' ends, addition of tails, shortening of the sequence, lengthening of the sequence, shifting of the sequence forward or backward by a few bases, or any combination thereof.
- Base modifications include, for example, 3'P, 5'P, 5-nitroindole, 2-aminopurine, 8-amino-2'-deoxyadenosine, C-5 propynyl-deoxycytidine, C-5 propynyl-deoxyuridine, 2-amino-2'-deoxyadenosine-5'-triphosphate, 2, 6-diaminopurine (2-amino-dA) , reverse dT, reverse dideoxy-T, hydroxymethyl dC, iso-dC, 5-methyl dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNA's) , and include at least one mismatched base at one of the bases, or a replacement of at least one of the bases with an RNA base, so as to achieve, for example, increasing nucleic acid interaction at the 3' end of the mutant-specific primer to increase Tm. Addition of double-stranded stabilizing base modifications has a positive effect on PCR, allowing PCR to be performed at a higher temperature, where Taq polymerase is known to show maximum activity. The modified probe should retain the ability to distinguish a mutant site to be detected from a wild-type site.
- The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented therefrom. It should be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the present invention, rather than to limit the present invention.
- Example 1. Primers and Probes Used in the Invention
- The primers and probes used in the invention are as shown in Tables 1-4:
- Table 1
-
- wherein the fluorescent group of NY-P is FAM; the fluorescent group of HV-P is HEX; the fluorescent group of PH-P is CY5; and the fluorescent gene of N-P is ROX.
- Table 2
-
-
- wherein the fluorescent group of NY-P is FAM; the fluorescent group of HV-P is HEX; the fluorescent group of PH-P is CY5; and the fluorescent gene of N-P is ROX.
- Table 3
-
- wherein the fluorophore of N501Y-P and E484K-P is FAM; the fluorophore of HV69-70Del-P and K417N-P is HEX; the fluorophore of P681H-P is CY5, the internal standard N-P probe of the novel coronavirus is labeled as a ROX channel, and the human gene internal standard IC-P probe is labeled as a CY5 channel.
- Table 4
-
-
- wherein L452R-P, P681R-P, L452Q-P, F490S-P and T478K-P probes are labeled as FAM channels, E484Q-P, E484K-P, T76I-P, Del247-253-P and D950N-P probes are labeled as HEX channels, the internal standard N-P probe of novel coronavirus is labeled as a ROX channel, and the human gene internal standard IC-P probe is labeled as a CY5 channel.
- Example 2. Method for Detecting Novel Coronavirus Variants
- A detection samples in the present invention was a throat swab, sputum, a bronchoalveolar lavage fluid, or blood. Viral nucleic acids were extracted by a magnetic bead method (a kit from Sansure Biotech INC. ) , and the following operations were performed in a sample treatment chamber:
- 2.1 an appropriate number of 1.5 mL sterilized centrifuge tubes were taken, and labeled as a negative control, a positive control and a testing sample, respectively, 300 μL of an RNA extraction solution 1 was added to each tube.
- 2.2 200 μL of the testing sample or the negative control or the positive control was added to each tube. The tube was covered with a cap, and shaken for 10 seconds for thorough mixing, and subjected to instant centrifugation.
- 2.3 100 μL of an RNA extraction solution 2-mix was added to each tube (sucked up after thorough mixing) , and the tube was shaken for 10 seconds for thorough mixing, and left to stand for 10 minutes at room temperature.
- 2.4 After instant centrifugation, the centrifuged tubes were placed on a separator, and the solution was slowly sucked out after 3 minutes (being careful not to touch a brown substance adhered to the tube wall) .
- 2.5 600 μL of an RNA extraction solution 3 and 200 μL of an RNA extraction solution 4 were added to each tube, and the tubes were shaken for 5 seconds for thorough mixing, and subjected to instant centrifugation, and the centrifuged tubes were placed on the separator again.
- 2.6 After about 3 minutes, the supernatant was divided into two layers. A pipette was inserted into the bottom of the centrifuged tubes, the liquid was slowly drawn from the bottom and discarded, and after standing for 1 minute, the residual liquid at the bottom of the tube was completely drawn out and discarded.
- 2.7 50 μL of PCR-mix was added to each tube, and a pipette was used to suck up the PCR-mix to elute the brown residue adhered to the wall of the centrifuged tube. The operation was repeated several times to elute the residue as completely as possible, and then all the eluted brown mixture was transferred to a 0.2mL PCR reaction tube, which was then covered with a cap and transferred to an amplification detection zone.
- The real-time fluorescent PCR reaction system was formulated as follows:
-
Components Volume/concentration in each reaction Mg 2+ 4 mM dNTPs (100 mM) 0.25 mM MMLV (10 U/μl) 10 U Taq enzyme (5 U/μl) 5 U Primer 100 nM Probe 50 nM PCR Buffer (1.5x) up to 50 μl - The PCR amplification procedure was set up as follows:
-
- Results analysis:
- 1) The target detection signals were FAM, HEX (or VIC) and ROX and the internal reference detection signal was CY5/ROX; and
- 2) Baseline setting: the baseline was generally set to 3-15 cycles, depending on the actual situations. The adjustment principle was: selecting a zone with a relatively sTable fluorescence signal before exponential amplification, to enable a starting point (Start) to avoid signal fluctuation during an initial stage of fluorescence acquisition, and to allow an end point (End) with cycles 1-2 less than Ct of the earliest exponentially amplified sample. Threshold setting: the setting principle was to make the threshold line just exceed the highest point of the normal negative control.
- 3) Results reading:
- Read the results for different compositions according to the following different rules:
- Read Rule 1
-
-
- Read Rule 2
-
-
-
-
- Example 3. Detection results of detecting the positive control by the composition of the present invention
- By using the compositions in Table 3 of the present invention and according to the method described in example 2, clinical novel coronavirus mutation samples (positive) from the First Hospital of Changsha, Hunan Province were subjected to fluorescence quantitative PCR to detect several major mutation sites for novel coronavirus nucleic acids, including mutation sites such as E484K, K417N, P681H, N501Y and HV69-70del, one of the samples was found to have typical mutations such as N501Y, HV69-70del and P681H (figures 1-2) , and one sample was also found to have E484 and K417N mutations (figures 3-4) . It can be seen from the figures that corresponding targets can be detected by the composition of the present invention according to determination rule 1, proving that the composition of the present invention can be used to detect novel coronavirus variants.
- By using the compositions in Table 4 of the present invention and according to the method described in example 2, a full-length transferred RNA was detected for the wild-type novel coronavirus, the alpha variant, the delta variant and the lambda variant. PCR detection was performed by a Hongshi fluorescent quantitative PCR instrument, and the results are shown in figures 5-11. It can be seen from the figures that corresponding targets can be detected by the composition of the present invention according to determination rule 2, proving that the composition of the present invention can be used to detect novel coronavirus variants.
- By using the compositions in Tables 1 and 2 of the present invention and according to the method described in example 2, samples (positive samples: artificially synthesized nucleic acid sequences containing N501Y, HV69-70del and P681H mutations; and negative samples: nucleic acids with no N501Y, HV69 -70del and P681H mutations extracted from inactivated novel coronavirus, and concentration calibration was performed by novel coronavirus nucleic acid detection kits) were detected, and the results are shown in figures 12-15. It can be seen from the figures that corresponding targets can be detected, proving that the composition of the present invention can be used to detect novel coronavirus variants.
- Example 4. Sensitivity Experiment for the Inventive Composition
- By using the compositions in Tables 1-4 of the present invention and according to the method described in example 2, detection was performed at 5 concentrations, 200,000, 20,000, 20,000, 20,000, 200, and 20 copies/ml of each target pseudovirus. Multiple PCR tests were performed by a Hongshi fluorescent quantitative PCR instrument, and the results are shown in figures 16-17 (compositions in Tables 1 and 2) , 18-20 (compositions in Table 3) , and 21-22 (compositions in Table 4) . It can be seen from the figures that corresponding targets can still be detected at a concentration as low as 200 copies/ml, proving that the sensitivity of the composition of the present invention is 200 copies/ml.
- Example 5. Specificity Experiment for the Inventive Composition
- By using the compositions of Tables 1-4 of the present invention and according to the method described in example 2, multiple PCR test were performed by a Hongshi fluorescent quantitative PCR instrument for pathogens which have homologous nucleic acid sequences and are prone to cause the same or similar clinical symptoms (such as coronaviruses (NL63, HKU1, 229E and 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. ) , and mutation detection results of all samples were negative. Some results are shown in figures 23-24 (figure 23 for the compositions in Table 3 and figure 24 for the compositions in Table 4) . It can be seen from the figure that the test results are all negative, and only the internal standard gene in the human genome was amplified. The compositions of the present invention are proved to have good specificity.
- Example 6. Accuracy Experiment for the Inventive Composition
- By using the compositions in Tables 3-4 of the present invention and according to the method described in example 2, strong-positive and weak-positive quality control products of 2 concentration levels (100 000 copies/ml and 2 000 copies/ml, respectively) were selected to measure the intra-batch accuracy and inter-batch accuracy, with 10 duplicate tests for each sample. The results show that detection rates of strong-positive and weak-positive reference products are both 100%, and the coefficient of variation (CV) for detected intra-batch and inter-batch Ct values is less than 5%, as shown in figures 25-28 (figures 25-26 for compositions in Table 3 and figures 27-28 for compositions in Table 4) . It is thus indicated that this kit has good intra-batch accuracy and inter-batch accuracy.
- Comparative Example 1. Other Primers and Probes with Poor Effect Designed By the Present Invention
- Due to the principle of complementary base pairing, dimers can be formed by primers and/or probes, but this probability is low and can be ruled out at the beginning of the design. However, when multiple pathogens are jointly detected, there are numerous primers and probes, and dimers are prone to form between a primer and a primer, a probe and a probe, or a primer and a probe. In order to ensure the conservation of the design (conservation is critical to the accuracy of detection) and take mutual interference between primers and probes into consideration, the primers and probes need to be carefully designed.
- Therefore, the inventor also designed other primers and probes (sequences not shown) to form different detection systems 1-4 (comparative examples to compositions in Table 3) , and 5-7 (comparative examples to compositions in Table 4) , which were also used to detect novel coronavirus mutations. Specific detection results are shown in figures 29-32 (comparative examples to compositions in Table 3) and 33-35 (comparative examples to compositions in Table 4) . It can be seen from the figures that the detection effects are poor.
Claims (16)
- A composition which can be used to detect major mutation sites of SARS-CoV-2 variants and identify the mutant types by combining detection results for different sites, the composition simultaneously comprising:a primer and probe combination for detecting mutation N501Y, a primer and probe combination for detecting mutation P681H, and a primer and probe combination for detecting mutation HV69-70del;wherein the primer and probe combination for detecting mutation N501Y is selected from one or more of the following groups:a primer and a probe shown by SEQ ID NO: 1-3, a primer and a probe shown by SEQ ID NO: 4-6, and a primer and a probe shown by SEQ ID NO: 7-9;wherein the primer and probe combination for detecting mutation P681H is selected from one or more of the following groups:a primer and a probe shown by SEQ ID NO: 10-12, a primer and a probe shown by SEQ ID NO: 13-15, and a primer and a probe shown by SEQ ID NO: 16-18;wherein, the primer and probe combination for detecting mutation HV69-70del is selected from one or more of the following groups:a primer and a probe shown by SEQ ID NO: 19-21, a primer and a probe shown by SEQ ID NO: 22-24, and a primer and a probe shown by SEQ ID NO: 25-27.
- The composition according to claim 1, wherein the composition simultaneously comprises:a) and/or b) :a) the primer and probe shown by SEQ ID NO: 1-3, the primer and probe shown by SEQ ID NO: 10-12, and the primer and probe shown by SEQ ID NO: 19-21; andb) the primer and probe shown by SEQ ID NO: 4-6, the primer and probe shown by SEQ ID NO: 13-15, and the primer and probe shown by SEQ ID NO: 22-24;
- The composition according to claim 1, wherein the composition simultaneously comprises:the primer and probe shown by SEQ ID NO: 7-9, the primer and probe shown by SEQ ID NO: 16-18, and the primer and probe shown by SEQ ID NO: 25-27.
- The composition according to any one of claims 1-3, wherein the composition further comprises:a primer and probe combination for detecting mutation K417N and a primer and probe combination for detecting mutation E484K;wherein the primer and probe combination for detecting mutation K417N is a primer and a probe shown by SEQ ID NO: 28-30; andwherein the primer and probe combination for detecting mutation E484K is a primer and a probe shown by SEQ ID NO: 31-33.
- The composition according to claim 4, wherein the composition includesa first nucleic acid composition:a mutant N501Y forward primer as shown by SEQ ID NO: 7, a mutant N501Y reverse primer as shown by SEQ ID NO: 8, and a mutant N501Y probe as shown by SEQ ID NO: 9; anda mutant HV69-70del forward primer shown by SEQ ID NO: 25, a mutant HV69-70del reverse primer shown by SEQ ID NO: 26, and a mutant HV69-70del probe shown by SEQ ID NO: 27; anda second nucleic acid composition:a mutant K417N forward primer as shown by SEQ ID NO: 28, a mutant K417N reverse primer as shown by SEQ ID NO: 29, and a mutant K417N probe as shown by SEQ ID NO: 30;a mutant E484K forward primer as shown by SEQ ID NO: 31, a mutant E484K reverse primer as shown by SEQ ID NO: 32, and a mutant E484K probe as shown by SEQ ID NO: 33; anda mutant P681H forward primer as shown by SEQ ID NO: 16, a mutant P681H reverse primer as shown by SEQ ID NO: 17, and a mutant P681H probe as shown by SEQ ID NO: 18.
- A composition which can be used to detect major mutation sites of SARS-CoV-2 variants and identify the mutant types by combining detection results for different sites, the composition simultaneously comprising:a first nucleic acid composition:a mutant L452R forward primer as shown by SEQ ID NO: 40, a mutant L452R reverse primer as shown by SEQ ID NO: 41, and a mutant L452R probe as shown by SEQ ID NO: 42; anda mutant E484Q forward primer as shown by SEQ ID NO: 43, a mutant E484Q reverse primer as shown by SEQ ID NO: 44, and a mutant E484Q probe as shown by SEQ ID NO: 45;a second nucleic acid composition:a mutant P681R forward primer as shown by SEQ ID NO: 46, a mutant P681R reverse primer as shown by SEQ ID NO: 47, and a mutant P681R probe as shown by SEQ ID NO: 48; anda mutant E484K forward primer as shown by SEQ ID NO: 49, a mutant E484K reverse primer as shown by SEQ ID NO: 50, and a mutant E484K probe as shown by SEQ ID NO: 51;a third nucleic acid composition:a mutant L452Q forward primer as shown by SEQ ID NO: 52, a mutant L452QR reverse primer as shown by SEQ ID NO: 53, and a mutant L452Q probe as shown by SEQ ID NO: 54; anda mutant T76I forward primer as shown by SEQ ID NO: 55, a mutant T76I reverse primer as shown by SEQ ID NO: 56, and a mutant T76I probe as shown by SEQ ID NO: 57;a fourth nucleic acid composition:a mutant F490S forward primer as shown by SEQ ID NO: 58, a mutant F490S reverse primer as shown by SEQ ID NO: 59, and a mutant F490S probe as shown by SEQ ID NO: 60; anda mutant Del247-253 forward primer as shown by SEQ ID NO: 61, a mutant Del247-253 reverse primer as shown by SEQ ID NO: 62, and a mutant Del247-253 probe as shown by SEQ ID NO: 63; anda fifth nucleic acid composition:a mutant T487K forward primer as shown by SEQ ID NO: 64, a mutant T487K reverse primer as shown by SEQ ID NO: 65, and a mutant T487K probe as shown by SEQ ID NO: 66; anda mutant D950N forward primer as shown by SEQ ID NO: 67, a mutant D950N reverse primer as shown by SEQ ID NO: 68, and a mutant D950N probe as shown by SEQ ID NO: 69.
- The composition according to any one of claims 1-6, wherein the composition further comprises an internal standard forward primer, an internal standard reverse primer, and an internal standard probe for monitoring.
- The composition according to claim 7, wherein the internal standard includes at least one of a human genome internal standard and a novel coronavirus internal standard.
- The composition according to claim 8, wherein the human genome internal standard comprises a human genome internal standard forward primer as shown by SEQ ID NO: 34, a human genome internal standard reverse primer as shown by SEQ ID NO: 35, and a human genome internal standard probe as shown by SEQ ID NO: 36.
- The composition according to claim 8, wherein the novel coronavirus internal standard comprises a novel coronavirus internal standard forward primer as shown by SEQ ID NO: 37, a novel coronavirus internal standard reverse primer as shown by SEQ ID NO: 38, and a novel coronavirus internal standard probe as shown by SEQ ID NO: 39; ora novel coronavirus internal standard forward primer as shown by SEQ ID NO: 70, a novel coronavirus internal standard reverse primer as shown by SEQ ID NO: 71, and a novel coronavirus internal standard probe as shown by SEQ ID NO: 72.
- The composition according to any one of claims 1-10, wherein the components of the composition are present in a mixed form.
- A use of the composition according to any one of claims 1-11 in the preparation of a kit for detecting major mutation sites of SARS-CoV-2 variants.
- A kit for detecting major mutation sites of SARS-CoV-2 variants, the kit comprising the composition of any one of claims 1-11.
- The kit according to claim 13, wherein the kit further comprises a nucleic acid release system and a nucleic acid amplification system.
- The kit according to claim 13, wherein the kit further comprises at least one of a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, a reverse transcriptase, a uracil glycosylase, a DNA polymerase, a PCR buffer, and Mg 2+.
- A method for detecting major mutation sites of SARS-CoV-2 variants, the method comprising the steps of:1) extracting or releasing nucleic acids in a sample to be tested;2) performing fluorescence quantitative PCR analysis on the nucleic acids obtained in step 1) by using the composition according to any one of claims 1-11; and3) obtaining and analyzing results.
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CN202110592489.6A CN115404237B (en) | 2021-05-28 | 2021-05-28 | Combination product, kit, use and method for detecting mutant SARS-CoV-2 virus |
CN202111500433 | 2021-12-09 | ||
CN202111503790.1A CN116254367A (en) | 2021-12-10 | 2021-12-10 | Compositions, kits, methods and uses for detecting SARS-CoV-2 mutation |
CN202111667275.7A CN114561490B (en) | 2021-12-09 | 2021-12-31 | Composition, kit and method for detecting SARS-CoV-2 mutation site and application thereof |
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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CN114107572B (en) * | 2022-01-26 | 2022-04-12 | 潮州凯普生物化学有限公司 | Primer probe set for detecting different new coronavirus mutant strains based on multiplex PCR technology, detection kit and application of detection kit |
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