CN114317831B - Kit for detecting novel coronavirus Omicron mutant strain - Google Patents
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- CN114317831B CN114317831B CN202210094440.2A CN202210094440A CN114317831B CN 114317831 B CN114317831 B CN 114317831B CN 202210094440 A CN202210094440 A CN 202210094440A CN 114317831 B CN114317831 B CN 114317831B
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- 241000711573 Coronaviridae Species 0.000 title claims abstract description 34
- 230000003321 amplification Effects 0.000 claims abstract description 27
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- 238000001514 detection method Methods 0.000 claims abstract description 20
- 238000009396 hybridization Methods 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 19
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- 238000004925 denaturation Methods 0.000 claims description 6
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- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
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- 239000003814 drug Substances 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 2
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- 102100031673 Corneodesmosin Human genes 0.000 abstract description 8
- 101710139375 Corneodesmosin Proteins 0.000 abstract description 8
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- 241000700605 Viruses Species 0.000 abstract description 3
- 238000012408 PCR amplification Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 9
- 230000000087 stabilizing effect Effects 0.000 description 7
- 108010006785 Taq Polymerase Proteins 0.000 description 6
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for detecting novel coronavirus Omicron mutant strain, belonging to the field of virus detection. The invention discloses a primer group and a molecular beacon for detecting novel coronavirus Omicron mutant, wherein the primer group comprises a novel coronavirus S protein Omicron mutant specific recognition primer pair and a stable primer, and can specifically amplify S protein Omicron mutant fragments; meanwhile, a specific recognition molecular beacon is designed in an amplification region defined by the primer pair and is used for detecting PCR amplification products; the invention provides a PCR technology based on specific mutation sites of an Omicron mutant strain, a primer group and a molecular beacon for detecting the Omicron mutant strain by molecular beacon hybridization, and constructs a kit and a detection method, and has the advantages of high sensitivity, strong identification capability, convenient operation, high detection speed, accurate result and the like, and is suitable for large-scale clinical application.
Description
Technical Field
The invention relates to the field of virus detection, in particular to a kit for detecting novel coronavirus Omicron mutant strain.
Background
Omicron mutant strains have higher transmission and infection capacity than wild-type viruses and bring about changes in clinical characteristics. These changes also present new challenges to current diagnostics, therapeutics, and vaccines. Therefore, the rapid identification of the novel coronavirus S protein coding genotype is not only beneficial to clinical diagnosis and treatment, but also beneficial to prevention and control of epidemic situation.
The current methods for identification of novel coronavirus mutants are mainly DNA sequencing techniques. However, this method has significant drawbacks: DNA sequencing techniques, while accurate, are time consuming, expensive, and not suitable for rapid detection requirements. There is a great clinical need for a means of detecting novel coronavirus mutants that is accurate, highly sensitive and simple to operate. ARMS-PCR (mutation amplification System) is a new technology developed on the basic PCR technology, the basic design concept is that a specific mutation site is placed at the 3 'end of a primer instead of the middle position, and the characteristic that heat-resistant Taq DNA polymerase lacks 3'. Fwdarw.5 'exonuclease activity is utilized, if a mismatch is formed on a 3' end base pair in the complementary pairing process of the primer and a template, a chain extension reaction is blocked due to the formation of a 3',5' -phosphodiester bond, and the amplification result is that: the amplification of wild type template is blocked, and the amplification of mutant type template product is performed, so that the method can be used for SNP typing detection. In view of the existence of novel coronavirus omacron at present, it is very necessary to provide a rapid, accurate and highly sensitive novel coronavirus mutant identification method.
Disclosure of Invention
The invention aims to provide a kit for detecting novel coronavirus Omicron mutant, which solves the problems existing in the prior art, comprises 3 pairs of novel coronavirus S protein Omicron mutant specific recognition primers and a molecular beacon, is used for detecting novel coronavirus mutant based on PCR technology and molecular beacon hybridization, has the advantages of high sensitivity, strong recognition capability, convenient operation, high detection speed, accurate results and the like, and is suitable for large-scale clinical application.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a primer set and a molecular beacon for detecting novel coronavirus Omicron mutant strain, wherein the primer set comprises:
NL211-212I INS214EPE_F:TTATAGTGCGTGAGCCAGAAGAT；
NL211-212I INS214EPE_R:GCTGTCCAACCTGAAGAAG；
NL211-212I INS214EPE_stable:CTCAGGGTTTTTCGGCTTTAGAAC；
S371LS373PS375F_F:CTATATAATCTCGCACCATTTTTC；
S371LS373PS375F_R:CTGACTTCATCACCTCTAAT；
S371LS373PS375F_stable:AAGTGTTATGGAGTGTCTCCTACT；
Q493RG496SQ498R_F:CTTTACGATCATATAGTTTTCG；
Q493RG496SQ498R_R:TGGTGCATGTAGAAGTTC；
the molecular beacon includes:
NL211-212I INS214EPE_B:
cgcaccGGTAGATTTGCCAATAGGTATTAACATCACTAGGggtgcg；
S371LS373PS375F_B:
cgcaccGATCTCTGCTTTACTAATGTCTATGCAGATTCggtgcg；
Q493RG496SQ498R_B:
cgcaccGGTGTTGGTCACCAACCATACAGAGggtgcg。
the invention also provides a product for detecting the novel coronavirus Omicron mutant strain, which comprises the primer group and a molecular beacon.
Preferably, the product comprises a kit, reagent or detection chip.
The invention also provides application of the primer group and the molecular beacon in preparing a product for detecting the novel coronavirus Omicron mutant strain.
Preferably, the product comprises a drug, kit, reagent or chip.
Preferably, the DNA of the sample to be detected is used as a template, the DNA is amplified by the primer set, and the amplified product is detected by hybridization with the molecular beacon, thereby determining whether the sample to be detected is a novel coronavirus Omicron mutant.
Preferably, the amplification system comprises the following components: hotStart Taq DNA polymerase 0.4. Mu.L, 5 Xreaction buffer 4. Mu.L, dNTP mix 1. Mu.L, primer F0.4. Mu.L, primer R0.4. Mu.L, primer stable 0.4. Mu.L, molecular beacon primer 0.2. Mu.L, template DNA 1. Mu.L, and enzyme-free water 12.2. Mu.L.
Preferably, the amplification procedure is: pre-denaturation at 95℃for 3 min; denaturation at 95℃for 5s, annealing at 60℃for 30s, and fluorescence signal acquisition, cycle 45 times.
The invention discloses the following technical effects:
the invention relates to a novel coronavirus Omicron mutant strain detection kit based on an ARMS-PCR technology and a molecular beacon technology, which comprises 3 groups of novel coronavirus S protein Omicron mutant strain specific recognition primers, and S protein Omicron mutant strain fragments can be specifically amplified by adopting the primer pair. Meanwhile, a specific recognition molecular beacon is designed in an amplification region defined by the primer pair for detecting the PCR product. The invention detects the characteristics of novel Omicron mutant strain of coronavirus: the mutant strain is amplified by using the mutant strain specific amplification primer, and the amplified product of the mutant strain is detected by molecular beacon hybridization, compared with the prior sequencing technology, the method has the advantages that: 1) The mutant strain fragments can be rapidly amplified through PCR, and the rapid detection of the combined molecular beacon is low in cost and does not need to carry out DNA sequencing; 2) The primer and the molecular beacon have the characteristics of accuracy and high definition; 3) The operation is simple and convenient, the speed is high, and the method is applicable to large-scale application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows amplification curves and Ct values for mutant specific primers of mutation sites NL211-212I INS214 EPE; a: amplification curves of different copy numbers Omicron mutant S genes by using NL211-212I INS214EPE mutant specific primers; b: amplification curves of different copy number wild type S genes amplified by NL211-212I INS214EPE mutant specific primers; c: ct values of A and B amplification curves;
FIG. 2 shows amplification curves and Ct values of specific primers for the mutation site S371LS373PS 375F; a: amplification curves of S371LS373PS375F mutant specific primers for amplifying different copy numbers Omicron mutant S genes; b: amplification curves of S371LS373PS375F mutant specific primers for amplifying wild type S genes with different copy numbers; c: ct values of A and B amplification curves;
FIG. 3 shows amplification curves and Ct values for mutant specific primers at mutation site Q493RG496SQ 498R; a: amplification curves of Q493RG496SQ498R mutant specific primers for amplifying different copy numbers Omicron mutant S genes; b: amplification curves of different copy numbers of wild type S genes amplified by Q493RG496SQ498R mutant specific primers; c: ct values of A and B amplification curves;
FIG. 4 shows amplification curves of NL211-212I INS214EPE mutant specific primer with/without stabilizing primer, respectively;
FIG. 5 shows the amplification curves of S371LS373PS375F mutant specific primers with/without stabilizing primers, respectively.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1 primers and molecular beacons for detection of novel coronavirus Omicron mutant
1. Primer design
(1) A pair of specific identification primer pairs and a stabilizing primer are respectively designed aiming at the novel coronavirus Omicron mutant S protein mutation sites NL211-212IINS214EPE, S371LS373PS375F and Q493RG496 SQ49RR, and the sequences are as follows:
TABLE 1 specific discrimination primer sequences
TABLE 2 Stable primers
The stable primer is positioned in an Omicron mutant primer amplicon region, is positioned between an Omicron forward primer and a molecular beacon binding region, comprises 3 '-end amino modification, can be bound to a template after high-temperature denaturation and annealing, forms relatively stable binding, and cannot be amplified after being bound due to the amino modification of the 3' -end. The stable state avoids the complex structure of the primer binding region, and can effectively improve the detection sensitivity of the Omicron mutant. After primer binding, the stable primer can be excised by 5 '. Fwdarw.3' exonuclease activity of Taq DNA polymerase without affecting subsequent PCR.
(2) DNA of the novel coronavirus Omicron mutant was extracted and amplified using the same as a template using the following amplification conditions:
the 20. Mu.L reaction system contained the following components: hotStart Taq DNA polymerase 0.4. Mu.L, 5 Xreaction buffer 4. Mu.L, dNTP mix (2.5 mM) 1. Mu.L, primer F0.4. Mu.L (10. Mu.M), primer R0.4. Mu.L (10. Mu.M), stabilizing primer 0.4. Mu.L (10. Mu.M), molecular beacon primer 0.2. Mu.L (10. Mu.M), template DNA 1. Mu.L, and enzyme-free water 12.2. Mu.L.
PCR reaction procedure: pre-denaturation at 95℃for 3 min; denaturation at 95℃for 5s, annealing at 60℃for 30s, and circulation 45 times, and detection of fluorescent signals was performed by using a Bio-rad CFX96 fluorescent quantitative PCR instrument.
2. For each pair of omacron mutant primers, the invention provides a molecular beacon probe for detecting the PCR product described above. The sequence is as follows:
TABLE 3 molecular beacon probes
The molecular beacon comprises: 5 '-cgcacccxxxxxxggtggcg-3' complementarily paired stem regions; specifically recognizing the circular region sequence of omicron mutant PCR product, the length of the sequence is 18-33bp, and the Tm value is about 70 ℃; the molecular beacon is modified by adopting a FAM fluorescent group at the 5 'end and a Dabcyl quenching group at the 3' end. In a natural state, the stem regions in the molecular beacons are complementarily paired, and the luminescent groups are close to the quenching groups, so that fluorescence emitted by the fluorescent groups is quenched by the quenching groups.
3. After the PCR system is uniformly mixed with a corresponding molecular beacon, the cyclic region in the molecular beacon can be hybridized with a specific PCR product through high-temperature denaturation and annealing to generate specific binding, so that the distance between a luminescent group and a quenching group is increased, and a corresponding optical signal is generated, namely the Omicron mutant fragment is amplified.
Example 2 novel Rapid detection kit for coronavirus Omicron mutant based on ARMS-PCR technology and molecular beacon technology
1. Novel rapid detection kit for omacron mutant strain of coronavirus comprises the following components:
1) Specific discrimination primers and stabilizing primers:
NL211-212I INS214EPE_F(SEQ ID NO：1):
TTATAGTGCGTGAGCCAGAAGAT；
NL211-212I INS214EPE_R(SEQ ID NO：2):
GCTGTCCAACCTGAAGAAG；
NL211-212I INS214EPE_stable(SEQ ID NO：3):
CTCAGGGTTTTTCGGCTTTAGAAC；
S371LS373PS375F_F(SEQ ID NO：4):
CTATATAATCTCGCACCATTTTTC；
S371LS373PS375F_R(SEQ ID NO：5):CTGACTTCATCACCTCTAAT；
S371LS373PS375F_stable(SEQ ID NO：6):
AAGTGTTATGGAGTGTCTCCTACT；
Q493RG496SQ498R_F(SEQ ID NO：7):
CTTTACGATCATATAGTTTTCG；
Q493RG496SQ498R_R(SEQ ID NO：8):TGGTGCATGTAGAAGTTC。
2) PCR premix: hotStart Taq DNA polymerase, 5 Xreaction buffer, dNTP.
3) Molecular beacon:
NL211-212I INS214EPE_B(SEQ ID NO：9):
cgcaccGGTAGATTTGCCAATAGGTATTAACATCACTAGGggtgcg；
S371LS373PS375F_B(SEQ ID NO：10):
cgcaccGATCTCTGCTTTACTAATGTCTATGCAGATTCggtgcg；
Q493RG496SQ498R_B(SEQ ID NO：11):
cgcaccGGTGTTGGTCACCAACCATACAGAGggtgcg。
4) Positive control and negative control recombinant plasmid
5) RNase/DNase water
2. A novel rapid detection method for a coronavirus Omicron mutant strain based on ARMS-PCR technology and molecular beacon technology. The specific operation is as follows:
1) Preparation of positive control and negative control recombinant plasmids
According to the published OmicronS protein gene sequence, gene synthesis is carried out by Nanjing Jinsri, and the gene is constructed to a cloning vector.
2) PCR system
The 20. Mu.L reaction system comprises the following components: hotStart Taq DNA polymerase 0.4. Mu.L, 5 Xreaction buffer 4. Mu.L, dNTP mix (2.5 mM) 1. Mu.L, primer F0.4. Mu.L (10. Mu.M), primer R0.4. Mu.L (10. Mu.M), stabilizing primer 0.4. Mu.L (10. Mu.M), molecular beacon primer 0.2. Mu.L (10. Mu.M), template DNA 1. Mu.L, RNase/DNase water 12.2. Mu.L.
3) PCR reaction procedure:
pre-denaturation at 95℃for 3 min; denaturation at 95℃for 5s, annealing at 60℃for 30s, and cycling 45 times. The detection of the fluorescent signal was performed using a Bio-rad CFX96 fluorescent quantitative PCR instrument.
3. Results and analysis
As shown in FIGS. 1-3, the Omicron-specific primers NL211-212I, INS214EPE, S371LS373PS375F and Q493RG496SQ498R and their molecular beacons amplify the amplification curves and Ct values of the Omicron mutant and wild-type novel coronavirus S proteins. The results show that these three pairs of primers can specifically amplify samples of omacron mutant strain, whereas wild type samples are essentially devoid of fluorescent signal. Furthermore, three pairs of primers can distinguish between omacron mutants and wild type samples as low as 300 copies per milliliter for templates of different copy numbers. The results in FIGS. 4-5 also show that the addition of the stabilizing primers to the reaction system can effectively improve the detection sensitivity of the Omacron mutant.
The invention designs omacron mutant specific amplification primer by ARMS-PCR technology, combines the design of stable primer, and identifies novel coronavirus mutant strain type by molecular beacon hybridization detection means, and the method can detect nucleic acid samples with the concentration of as low as 300 copies per milliliter in 60 minutes. The method can detect by a fluorescent quantitative PCR instrument, does not need additional equipment and does not need sequencing identification.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Sequence listing
<110> Hangzhou medical college
<120> a kit for detecting novel coronavirus Omicron mutant
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Claims (7)
1. A primer set and a molecular beacon for detecting novel coronavirus omacron mutant, characterized in that the primer set comprises:
NL211-212I INS214EPE_F: TTATAGTGCGTGAGCCAGAAGAT；
NL211-212I INS214EPE_R: GCTGTCCAACCTGAAGAAG；
NL211-212I INS214EPE_stable: CTCAGGGTTTTTCGGCTTTAGAAC；
S371LS373PS375F_F: CTATATAATCTCGCACCATTTTTC；
S371LS373PS375F_R: CTGACTTCATCACCTCTAAT；
S371LS373PS375F_stable: AAGTGTTATGGAGTGTCTCCTACT；
Q493RG496SQ498R_F: CTTTACGATCATATAGTTTTCG；
Q493RG496SQ498R_R: TGGTGCATGTAGAAGTTC；
the molecular beacon includes:
NL211-212I INS214EPE_B:
cgcaccGGTAGATTTGCCAATAGGTATTAACATCACTAGGggtgcg；
S371LS373PS375F_B:
cgcaccGATCTCTGCTTTACTAATGTCTATGCAGATTCggtgcg；
Q493RG496SQ498R _B:
cgcaccGGTGTTGGTCACCAACCATACAGAGggtgcg。
2. a product for detecting novel coronavirus omacron mutants, comprising the primer set of claim 1 and a molecular beacon.
3. The product of claim 2, wherein the product comprises a kit, reagent or detection chip.
4. Use of the primer set of claim 1 and a molecular beacon for the preparation of a product for detecting novel coronavirus Omicron mutant.
5. The use of claim 4, wherein the product comprises a drug, a kit, a reagent or a chip.
6. The method according to claim 4, wherein the DNA of the sample to be detected is used as a template, the DNA is amplified by the primer set, and the amplified product is detected by hybridization of the molecular beacon, so as to determine whether the sample to be detected is a novel coronavirus Omicron mutant.
7. The use of claim 6, wherein the amplification procedure is: 95. pre-denaturation at 3 min; 95. denaturation at 5℃for 30s, annealing at 60℃for 30s, and fluorescence signal acquisition, cycle 45 times.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113308574A (en) * | 2021-06-01 | 2021-08-27 | 上海伯杰医疗科技有限公司 | Primer probe combination, kit and parting detection method for detecting novel coronavirus mutant strain |
| CN113943838A (en) * | 2021-12-09 | 2022-01-18 | 常州国药医学检验实验室有限公司 | New coronavirus Ormckh mutation sequence detection technology based on multiple fluorescence quantitative ARMS-PCR technology and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113308574A (en) * | 2021-06-01 | 2021-08-27 | 上海伯杰医疗科技有限公司 | Primer probe combination, kit and parting detection method for detecting novel coronavirus mutant strain |
| CN113943838A (en) * | 2021-12-09 | 2022-01-18 | 常州国药医学检验实验室有限公司 | New coronavirus Ormckh mutation sequence detection technology based on multiple fluorescence quantitative ARMS-PCR technology and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| S glycoprotein diversity of the Omicron variant;Rakesh Sarkar等;medRxiv;第1-30页 * |
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