CN115896351A - Genotyping detection method for mutation of new coronavirus S protein L452R - Google Patents
Genotyping detection method for mutation of new coronavirus S protein L452R Download PDFInfo
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Abstract
The invention relates to the technical field of virus genotyping detection, in particular to a genotyping detection method for mutation of S protein L452R of novel coronavirus. The RPA-PfAgo method established by the invention can be used for genotyping the key mutation L452R of the new coronavirus, and the method utilizes the specific recognition and cutting characteristics of PfAgo and realizes the high sensitivity of each reaction single copy, which is the highest sensitivity in theory and the highest sensitivity of the existing SARS-CoV-2 mutation detection. The method can be completed within 1.5 hours, only needs simple instrument support, and is convenient for field application. Meanwhile, it provides strong support for the precise control and precise medical treatment of SARS-CoV-2 mutation, and can easily realize the genotyping of multiple SARS-CoV-2 key mutations.
Description
Technical Field
The invention relates to the technical field of virus genotyping detection, in particular to a genotyping detection method for mutation of S protein L452R of novel coronavirus.
Background
The mutation of the new coronavirus (SARS-CoV-2) has caused one wave to another and the pandemic has not yet ended. The key mutations in the S protein often confer greater infectivity and immune escape capacity to the virus, which makes it difficult to prevent and control the epidemic. For example, the L452R-bearing variant allows SARS-CoV-2 to escape Human Leukocyte Antigen (HLA) -restricted cellular immunity, enhances the affinity of the virus for the receptor ACE2, and enhances infectivity and immune escape. Therefore, an effective genotyping method developed aiming at the key mutation L452R can provide help for precise prevention and control and precise treatment of epidemic situations. To date, the major genotyping method for the key mutations of the new coronavirus is the second generation sequencing technology (NGS). However, this method has certain limitations, such as being time consuming and labor intensive, relying on laboratory instrumentation, and the like. In recent years, restriction enzyme-based detection techniques have been widely used in the field of genotyping. Pfago is an Argonaute protein in Pyrococcus furiosus, mainly takes ssDNA phosphorylated at the 5' end as a target, and specifically cuts a complementary DNA sequence. By mismatch design of gDNA, single base discrimination of target nucleic acids can be achieved. Ruyi He et al established a PAND method based on this property of Pfago, which is the first application of Pfago to the field of molecular detection. The method combines PCR/tHDA and Pfago, realizes DNA detection with attomol sensitivity on a human breast cancer related gene BRAC1, can detect tumor mutants KRAS G12D and EGFR T790M at the same time, and detects 0.1% of mutation at the lowest. The detection method has the advantages of high sensitivity, strong specificity and the like, but relates to temperature control equipment and has longer reaction time. Therefore, the method can be optimized based on the method, and a simpler and more efficient mode is developed and applied to the genotyping detection of the mutation of the S protein L452R of the novel coronavirus.
Disclosure of Invention
The invention provides a new rapid, efficient and high-specificity molecular detection method for genotyping of the L452R gene of the S protein of the new coronavirus based on the RPA-PfAgo technology.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
in a first aspect of the present invention, a primer set is provided, which includes a primer pair for RPA amplification of a target gene L452R, and a guide DNA and a molecular beacon for PfAgo reaction, and the sequences are as follows:
RPA-F：CTAACAATCTTGATTCTAAGGTTGGTGG；
RPA-R：CTCTCTCAAAAGGTTTGAGATTAGA；
guide DNA CGGTATAGATTGTTTA;
molecular beacons: FAM-CGCACCAATTACCGGTATAGATGGTGCG-BHQ1.
In a second aspect of the invention, the application of the primer group is provided, which comprises genotyping a key mutation gene L452R of the new coronavirus.
In a third aspect of the invention, a kit is provided, which comprises the primer set, the RPA nucleic acid amplification lyophilized enzyme and the Pfago enzyme.
In a fourth aspect of the invention, the use of the kit is provided, which comprises genotyping the key mutation gene L452R of the novel coronavirus.
In a fifth aspect of the present invention, there is provided a method for genotyping a key mutant gene L452R of a novel coronavirus, comprising the steps of:
(1) Extracting nucleic acid in a sample to be detected, wherein the extracting solution is used as a template for RPA reaction;
(2) Adopting an RPA amplification primer pair to carry out RPA amplification on the nucleic acid of the sample to be detected;
(3) Mixing the guide DNA, the molecular beacon and the Pfago enzyme with the RPA amplification product obtained in the step (2) for enzyme digestion reaction;
(4) Detecting the fluorescence condition generated after the molecular beacon in the reaction system is cut, and if the fluorescence value is obviously different from that of a negative control, judging the L452R mutant product existing in the RPA product; if the fluorescence value is not significantly different from the negative control, the L452R mutant product is judged to be absent in the RPA product.
Preferably, the RPA reaction system in step (2) comprises the following components: mu.L of nucleic acid extract, 2. Mu.L of each of 10. Mu.M RPA-F and RPA-F primers.
Preferably, the RPA reaction conditions in step (2) are: the reaction was initiated by the addition of 2.5. Mu.L magnesium acetate at a concentration of 280mM and incubated at 42 ℃ for 30min.
Preferably, the PfAgo reaction system in step (3) comprises the following components: mu.L of RPA amplification product as a template, 2. Mu.L of Pfago protein at a concentration of 15. Mu.M, 2. Mu.L of guide DNA at a concentration of 20. Mu.M, 0.5. Mu.L of molecular beacon at a concentration of 20. Mu.M, 2. Mu.L of 1 × reaction Buffer, and 8.5. Mu.L of ultrapure water.
Preferably, the reaction conditions of Pfago in step (3) are: after mixing and centrifugation, the mixture is put into a fluorescence quantitative PCR instrument and fully reacted for 45 minutes at 95 ℃, and FAM fluorescence signals are recorded every 60 seconds.
The specific principle of the invention is as follows: the sample nucleic acid was amplified by RPA reaction, the specific sequence was digested with guide DNA and PfAgo protein, and whether or not the target sequence was contained was determined based on the intensity of the fluorescence intensity of the molecular beacon with respect to the control group (fig. 1).
The invention has the beneficial effects that: the RPA-PfAgo method established by the invention can be used for genotyping the key mutation L452R of the new coronavirus, and the method utilizes the specific recognition and cutting characteristics of PfAgo and realizes the high sensitivity of each reaction single copy, which is the highest sensitivity in theory and the highest sensitivity of the existing SARS-CoV-2 mutation detection. The method can be completed within 1.5 hours, only needs simple instrument support, and is convenient for field application. Meanwhile, it provides strong support for the precise control and precise medical treatment of SARS-CoV-2 mutation, and can easily realize the genotyping of multiple SARS-CoV-2 key mutations.
Correlation definition
RPA: recombinase polymerase isothermal nucleic acid amplification technology
L452R gene wild type: l452 (WT)
L452R gene mutant: r452 (MT)
Drawings
FIG. 1 schematic representation of the RPA-PfAgo process
FIG. 2 sensitivity reaction of the RPA-Pfago method SARS-CoV-2 Mutant (MT) and Wild Type (WT) RNA standards were used. The fluorescence curves (A) and the end-point fluorescence signals (B) of the reactions with different amounts of template of the RNA standard are shown. Error bars represent standard error of triplicate.
FIG. 3 validation of the RPA-PfAgo method with SARS-CoV-2-MT-B.1.617 pseudovirus: fluorescence curves (A) of different RNA amounts for reaction, end point fluorescence signals (B) and end point visualization images (C) under blue light are extracted from SARS-CoV-2 pseudovirus. Error bars represent standard error of triplicate. * P <0.001.
Detailed description of the preferred embodiments
The present invention is further described below with reference to specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Reagent and apparatus
TIANAmp Virus RNA Kit from Tiangen Biochemical technology (Beijing) Ltd; the T7 High Efficiency Transcription Kit is purchased from Beijing Quanyujin Biotechnology GmbH; the RT-RPA nucleic acid amplification kit is purchased from Hangzhou mass measurement biotechnology limited company; universal SYBR qPCR Master Mix, hiScript 1stStrand cDNA Synthesis Kit,
Gel DNA Extraction Mini Kit was purchased from Biotechnology, inc. of King of Nanjing NuoZan; SARS-CoV-2-MT-B.1.617 pseudovirus was purchased from Bai' ao (Suzhou) Biotechnology Ltd; the Qubit 4 fluorometer was purchased from siemer femier technologies (china) ltd; roche LightCycler 480II qPCR instrument was purchased from Switzerland.
Design of primers, guide DNA and molecular beacons
A fragment containing the L452R site on the SARS-CoV-2 Gene (GenBank accession No. NC-045512.2 22793-23430 nt) was selected in NCBI, RT-qPCR primers S Gene-Forward and S Gene-Reverse were designed, and the amplicon size was 638bp. A forward primer RPA-F and a reverse primer RPA-R are respectively designed before and after a SARS-CoV-2L452R mutation site according to the design principle of the RPA primer, so that the RPA amplicon has a length of 60-100bp. Guide DNA (gDNA 1, 1696t in length, phosphorylated at the 5' end) and molecular beacon MB1 were designed based on the mutation target. All oligonucleotides were synthesized by Anhui Universal Biopsis (Anhui, china), and the sequences are shown in Table 1.
TABLE 1 primer and Probe sequences
Example 1 construction of RNA standards
The L452R mutation site (T > G) is located at 22917nt of SARS-CoV-2 gene. A638 nt fragment (GenBank accession No. NC-045512.2, 22793-23430 nt) containing the mutation site was inserted between the T7 promoter and T7 terminator of pET-28b (+) vector to construct pET28b-S gene standard plasmid by DNA synthesis and molecular cloning. Using point mutations, a pair of wild type and mutant standard plasmids were constructed using T or G at the L452R mutation site. In vitro Transcription of RNA standards was performed according to the T7 High Efficiency Transcription Kit instructions using wild type and mutant standard plasmids as templates. After in vitro transcription, the RNA standards were purified by phenol-chloroform extraction, dissolved in DEPC water and stored at-80 ℃. RNA standards were quantified by a Qubit 4 fluorometer and the copy number calculated from their size (5971 bp). Using serially diluted standard plasmids, a qPCR standard curve of copy number versus Ct value was plotted and used for RT-qPCR determination of copy number of RNA standards.
Example 2 expression purification of Pfago
The gene sequence of Pfago was obtained from the literature (Chem Commun (Camb) 2019,55 (88), 13219-13222.) and was universally biosynthesized onto pET28b (+) vector (purchased by general biosystems, anhui, inc.) to obtain pET28b-6 XHis-Pfago with His tag at the N-terminus. The recombinant plasmid was transformed into escherichia coli BL21 (DE 3) pLyss chemically competent cells e.coli BL21 (DE 3) pLyss and purified by nickel affinity chromatography using AKTA Prime Plus system. The purified Pfago protein was analyzed by SDS-PAGE and after overnight dialysis it was stored in buffer at-80 ℃.
EXAMPLE 3RPA reaction System
The RPA amplification was performed in part according to the instructions for the RPA nucleic acid amplification reagents (Zhongzhou Mass Biotech, inc., china). In a 50. Mu.L reaction system, 41.5. Mu.L of A Buffer, 2. Mu.L each of primers RPA-F (10. Mu.M) and RPA-R (10. Mu.M), and 3. Mu.L each of the nucleic acid extracts were added to a detection unit tube containing the RPA nucleic acid amplification lyophylase. Next, 2.5. Mu.L of RPA Buffer B (containing 280mM magnesium acetate) was added to the cap of the detection unit to start the reaction, and after centrifugation, the reaction was incubated at 42 ℃ for 30min. After obtaining the RPA amplification product, the method is based on
Gel DNA Extraction Mini Kit instructions for column purification.
Example 4Pfago reaction System
In the experimental group, 2. Mu.L of Pfago protein (15. Mu.M), 2. Mu.L of guide DNA (20. Mu.M), 0.5. Mu.L of molecular beacon (20. Mu.M), 2. Mu.L of 1 × reaction Buffer, and 8.5. Mu.L of ultrapure water were sequentially added to the column-purified RPA amplification product of 5. Mu.L as a template, and the total amount was 20. Mu.L. Mixing, centrifuging, placing in Roche LightCycler 480II qPCR instrument, reacting at 95 deg.C for 45min, recording FAM fluorescence signal every 60 s, and replacing RPA amplification product with blank solvent in control group.
Example 5 sensitivity of the RPA-Pfago method
The virus load in the actual detection process affects the detection result, so the sensitivity of the method needs to be evaluated. In vitro transcribed wild type and mutant RNA standards were 10-fold diluted to 10 4 、10 2 、10 1 、10 0 copies, sensitivity was assessed using established methods. The results showed that the established RPA-Pfago method had a minimum detection limit of 1copies for the Mutant (MT) and no detection for the Wild Type (WT) (FIG. 2).
EXAMPLE 6 method of verifying pseudovirus by SARS-CoV-2-MT-B.1.617
By usingVerification of the method for SARS-CoV-2-MT-B.1.617 pseudovirus containing L452R mutation by extraction of RNA using the TIANAmp Virus RNA Kit and adjustment of the concentration to 10 4 、10 2 、10 1 、10 0 copies was detected with Control group of 10 4 copies SARS-CoV-2 wild type RNA standard. The results show that the RPA-PfAgo method detects the L452R mutation present in pseudoviruses at all four concentrations (FIG. 3).
Claims (9)
1. A primer group comprises a primer pair for carrying out RPA amplification on a target gene L452R, and a guide DNA and a molecular beacon of Pfago reaction, wherein the sequences are as follows:
RPA-F：CTAACAATCTTGATTCTAAGGTTGGTGG；
RPA-R：CTCTCTCAAAAGGTTTGAGATTAGA；
guide DN A CGGTATAGATTGTTTA;
molecular beacons: FAM-CGCACCAATTACCGGTATAGATGGTGCG-BHQ1.
2. Use of the primer set according to claim 1, comprising genotyping the essential mutant gene L452R of neocoronavirus.
3. A kit comprising the primer set of claim 1, RPA nucleic acid amplification lyophilisation enzyme, and PfAgo enzyme.
4. The use according to claim 3 in a kit comprising genotyping the essential mutant gene L452R of neocoronaviruse.
5. A method for genotyping a key mutant gene L452R of a novel coronavirus, comprising the following steps:
(1) Extracting nucleic acid in a sample to be detected, wherein the extracting solution is used as a template for RPA reaction;
(2) Adopting an RPA amplification primer pair to carry out RPA amplification on the nucleic acid of the sample to be detected;
(3) Mixing the guide DNA, the molecular beacon and the Pfago enzyme with the RPA amplification product obtained in the step (2) to perform enzyme digestion reaction;
(4) Detecting the fluorescence condition generated after the molecular beacon in the reaction system is cut, and if the fluorescence value is obviously different from that of a negative control, judging the L452R mutant product existing in the RPA product; if the fluorescence value is not significantly different from the negative control, the L452R mutant product is judged to be absent in the RPA product.
6. The method of claim 5, wherein the RPA reaction system in step (2) comprises the following components: mu.L of the nucleic acid extract, 2. Mu.L of each of 10. Mu.M RPA-F and RPA-F primers.
7. The method of claim 5, wherein the RPA reaction conditions in step (2) are: the reaction was initiated by the addition of 2.5. Mu.L magnesium acetate at a concentration of 280mM and incubated at 42 ℃ for 30min.
8. The method as claimed in claim 5, wherein the Pfago reaction system in the step (3) comprises the following components: mu.L of RPA amplification product as template, 2. Mu.L of Pfago protein at a concentration of 15. Mu.M, 2. Mu.L of guide DNA at a concentration of 20. Mu.M, 0.5. Mu.L of molecular beacon at a concentration of 20. Mu.M, 2. Mu.L of 1 × reaction Buffer, and 8.5. Mu.L of ultrapure water.
9. The method of claim 5, wherein the Pfago reaction conditions in step (3) are: after mixing and centrifugation, the mixture is put into a fluorescence quantitative PCR instrument and fully reacted for 45 minutes at 95 ℃, and FAM fluorescence signals are recorded every 60 seconds.
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Citations (5)
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|---|---|---|---|---|
| CN108103151A (en) * | 2017-12-08 | 2018-06-01 | 东南大学 | A kind of method and its application based on the protein-bonded detection of nucleic acids of sequence-specific nucleic acid and parting |
| WO2019192156A1 (en) * | 2018-04-03 | 2019-10-10 | 上海交通大学 | Method for detecting nucleic acid based on prokaryotic argonaute protein and application thereof |
| CN110643687A (en) * | 2019-11-19 | 2020-01-03 | 深圳市艾伟迪生物科技有限公司 | SRDA (sequence-related amplified deoxyribonucleic acid) isothermal nucleic acid amplification kit and application thereof |
| CN113215314A (en) * | 2021-05-10 | 2021-08-06 | 江苏海洋大学 | Probe, primer set, kit and detection method for rapidly detecting SARS-CoV-2 by using L/RPA |
| WO2022132955A2 (en) * | 2020-12-16 | 2022-06-23 | Proof Diagnostics, Inc. | Coronavirus rapid diagnostics |
-
2022
- 2022-11-17 CN CN202211439079.9A patent/CN115896351A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103151A (en) * | 2017-12-08 | 2018-06-01 | 东南大学 | A kind of method and its application based on the protein-bonded detection of nucleic acids of sequence-specific nucleic acid and parting |
| WO2019192156A1 (en) * | 2018-04-03 | 2019-10-10 | 上海交通大学 | Method for detecting nucleic acid based on prokaryotic argonaute protein and application thereof |
| CN110643687A (en) * | 2019-11-19 | 2020-01-03 | 深圳市艾伟迪生物科技有限公司 | SRDA (sequence-related amplified deoxyribonucleic acid) isothermal nucleic acid amplification kit and application thereof |
| WO2022132955A2 (en) * | 2020-12-16 | 2022-06-23 | Proof Diagnostics, Inc. | Coronavirus rapid diagnostics |
| CN113215314A (en) * | 2021-05-10 | 2021-08-06 | 江苏海洋大学 | Probe, primer set, kit and detection method for rapidly detecting SARS-CoV-2 by using L/RPA |
Non-Patent Citations (1)
| Title |
|---|
| YUQING QIN等: "Emerging Argonaute-based nucleic acid biosensors", 《TRENDS IN BIOTECHNOLOGY》, vol. 40, no. 8, 31 August 2022 (2022-08-31), pages 910 - 914, XP087118151, DOI: 10.1016/j.tibtech.2022.03.006 * |
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