CN114480742A - qRT-PCR method for identifying BA-1 branch of novel coronavirus Omicron variant - Google Patents
qRT-PCR method for identifying BA-1 branch of novel coronavirus Omicron variant Download PDFInfo
- Publication number
- CN114480742A CN114480742A CN202210187453.4A CN202210187453A CN114480742A CN 114480742 A CN114480742 A CN 114480742A CN 202210187453 A CN202210187453 A CN 202210187453A CN 114480742 A CN114480742 A CN 114480742A
- Authority
- CN
- China
- Prior art keywords
- seq
- primer
- variant
- mutation
- nucleotide sequence
- 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
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000011529 RT qPCR Methods 0.000 title claims abstract description 29
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 23
- 230000035772 mutation Effects 0.000 claims abstract description 71
- 239000000523 sample Substances 0.000 claims abstract description 51
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 49
- 238000001514 detection method Methods 0.000 claims abstract description 43
- 230000003321 amplification Effects 0.000 claims abstract description 20
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 20
- 230000003612 virological effect Effects 0.000 claims abstract description 9
- 239000002773 nucleotide Substances 0.000 claims description 36
- 125000003729 nucleotide group Chemical group 0.000 claims description 36
- 102220590697 Spindlin-1_A67V_mutation Human genes 0.000 claims description 9
- 238000013461 design Methods 0.000 claims description 9
- 238000002474 experimental method Methods 0.000 claims description 6
- 238000003753 real-time PCR Methods 0.000 claims description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 239000012295 chemical reaction liquid Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 108020004707 nucleic acids Proteins 0.000 abstract description 20
- 102000039446 nucleic acids Human genes 0.000 abstract description 20
- 150000007523 nucleic acids Chemical class 0.000 abstract description 20
- 230000035945 sensitivity Effects 0.000 abstract description 14
- 241000700605 Viruses Species 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 16
- 238000012163 sequencing technique Methods 0.000 description 12
- 238000012360 testing method Methods 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 230000004907 flux Effects 0.000 description 3
- 206010035664 Pneumonia Diseases 0.000 description 2
- 229940096437 Protein S Drugs 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 101710198474 Spike protein Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 1
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 102200110418 rs570878629 Human genes 0.000 description 1
Images
Classifications
-
- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to a qRT-PCR method for identifying branches of a novel coronavirus Omicron variant BA-1. In the method, in order to improve the detection sensitivity and specificity, a TaqMan probe is introduced; in order to reduce the cost, 2 pairs of primers are changed into 1 pair of half primers, namely: 2 upstream primers respectively target mutation sites and original non-mutation sites, and 1 downstream primer is shared by 2 upstream primers. By reducing the degree of matching between the variant primers and the non-variant viral nucleic acids and between the non-variant primers and the variant viral nucleic acids in the reaction system, the amplification curve for amplifying the variant viral nucleic acids by the variant primers in the reaction system is earlier than the amplification curve for amplifying the non-variant nucleic acids, and similarly, the amplification curve for amplifying the non-variant nucleic acids by the non-variant primers is earlier than the amplification curve for amplifying the variant nucleic acids.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a qRT-PCR method for identifying branches of a novel coronavirus Omicron variant BA-1.
Background
Gene sequencing is the most common new coronavirus variety identification technology at present, and the technology has the following disadvantages[1]: 1. the time from sample to result is long, and the uncontrollable influence factors are many. The time from sample processing to result reporting is 6-8 hours, and most virus detection units do not have gene sequencing conditions and need to be handed to a third-party sequencing company for generation and testing, and the sample road transportation and possible computer queuing waiting time lead the gene sequencing not only to be slow, but also to have a plurality of uncontrollable influence factors; 2. the detection sensitivity is low. Before the gene sequencing is tested on a computer, gene amplification is needed, and sequencing can be influenced if the amplification effect of a low-abundance nucleic acid sample is slightly poor; 3. the detection cost is high. The sequencing of the new coronavirus gene comprises complicated steps of sample nucleic acid extraction, RT-PCR, PCR product purification, on-machine testing and the like, so that the detection material and labor cost are high, and the deviation of the detection result is easily caused by careless operation in multi-step operation. The defects enable gene sequencing to influence the detection timeliness, flux and cost of virus variant strains when being applied to the prevention and control of the new coronary pneumonia epidemic situation, possibly influence the detection result and have important influence on the prevention and control work of the new coronary pneumonia epidemic situation[2]。
New coronavirus Omicron variant (also called B.1.1.529 branch) has 42 variants such as T19I in virus Spike protein[3]And has higher replication force and propagation force. Delta variant has been rapidly replaced in 2022 as the main new coronavirus epidemic variant in the world[3]The work of preventing and controlling the new crown epidemic situation needs a rapid detection method of the new crown virus Omicron variant strain. After the occurrence of the Omicron variant, the Omicron variant rapidly evolves into branches BA-1, BA.2, BA.3 and the like, has different etiological characteristics, and needs to monitor and control each branch respectively[4]. The branch of the Omicron variant BA-1 generates unique mutations such as A67V, Delta 143-145, N211I, Delta 211, 215EPEins, S371L, G446S, G496S, T547K, N856K and L981F in virus Spike protein, and can be used for distinguishing the branch BA-1 and BA.2 of the Omicron variant and the Omicron variant from Alpha, Beta, Gamma and Delta variants.
Reference documents:
[1]Bezerra et al,A Sanger-based approach for scaling up screening of SARS-CoV-2variants of interest and concern.Infection,Genetics and Evolution, 2021,92:104910.
[2]Vega-Magana,et al.RT-qPCR Assays for Rapid Detection of the N501Y, 69-70del,K417N,and E484K SARS-CoV-2Mutations:A Screening Strategy to Identify Variants with Clinical Impact.Frontiers in Cellular and Infection Microbiology,2021,11:672562.
[3]GowrisankarA,Priyanka TM,Banerjee S"Omicron:a mysterious variant of concern".The EuropeanPhysical Journal Plus,2022,137(1):100.
[4]Kumar S,Karuppanan K,Subramaniam G.Omicron(BA-1)and Sub-Variants(BA-1,BA.2and BA.3)of SARS-CoV-2Spike Infectivity and Pathogenicity:A Comparative Sequence and Structural-based Computational Assessment,bioRxiv,2022,doi:https://doi.org/10.1101/2022.02.11.480029.
disclosure of Invention
Aiming at the problems, the invention provides a qRT-PCR method for identifying the branch of a novel coronavirus Omicron variant BA-1.
In order to achieve the purpose, the invention adopts the following technical scheme:
by using an improved PCR method named as an Amplification Retardation Mutation System (ARMS), the virus variant can be accurately identified by using fluorescent quantitative PCR instruments equipped by most detection units through complicated operation steps of virus nucleic acid extraction-qRT-PCR (quantitative reverse transcription-polymerase chain reaction) instead of virus nucleic acid extraction-RT-PCR amplification-PCR product purification-on-machine test and the like of gene sequencing, the dependence on expensive sequencers is overcome, and the universality of the detection method of the new coronavirus variant is improved. The high-sensitivity, high-flux and low-cost detection of the new coronavirus variant is realized by a 96-hole or 384-hole qRT-PCR reaction plate and the high-sensitivity and low-cost point of the qRT-PCR by a TaqMan probe method.
The basic method technology system consists of 2 pairs of primers, wherein one pair of primers is combined with a mutation site, the other pair of primers is combined with an original non-mutation site, and whether the mutation exists is judged according to the existence of amplified fragments and the length of the fragments.
In the method, a TaqMan probe is introduced for improving the detection sensitivity and specificity; in order to reduce the cost, 2 pairs of primers are changed into 1 pair of half primers, namely: 2 upstream primers respectively target mutation sites and original non-mutation sites, and 1 downstream primer is shared by 2 upstream primers. By reducing the degree of matching between the variant primers and the non-variant viral nucleic acids and between the non-variant primers and the variant viral nucleic acids in the reaction system, the amplification curve for amplifying the variant viral nucleic acids by the variant primers in the reaction system is earlier than the amplification curve for amplifying the non-variant nucleic acids, and similarly, the amplification curve for amplifying the non-variant nucleic acids by the non-variant primers is earlier than the amplification curve for amplifying the variant nucleic acids. And (3) detecting the sample by using two sets of experimental systems of the variation primer and the non-variation primer, wherein the variation primer system is used for judging that the detection sample contains the variation virus when an amplification curve of the variation primer system is earlier than that of the non-variation primer.
A qRT-PCR method for identifying the branch of a novel coronavirus Omicron variant BA-1 comprises the following steps:
step 1, extracting new coronavirus RNA by using a Trizol method;
step 2.1, primer design: according to the general principle of primer design, combining nucleotide sequences near the variation sites of the novel coronavirus Indian variety, and designing two pairs of primers for each variation site, wherein the 3' end of an upstream primer is respectively matched with a variation point and a non-variation point, namely a variation upstream primer and a non-variation upstream primer, and the two pairs of upstream primers share a downstream primer;
when the mutation site to be identified is A67V, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 1: 5'-CCAATGTTACTTGGTTCCATGT-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 2: 5'-CCAATGTTACTTGGTTCCATGC-3', the downstream primer is shown in SEQ ID NO. 3: 5'-TGTTAGACTTCTCAGTGGAAGC-3', respectively;
when the variation site to be identified is N211I, the nucleotide sequence of the variation upstream primer is shown in SEQ ID NO. 4: 5'-AATATATTCTAAGCACACGCCTATTAT-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 5: 5'-AATATATTCTAAGCACACGCCTATTAA-3', the downstream primer is shown in SEQ ID NO. 6: 5'-GAGTCAAATAACTTCTATGTAAAGCA AG-3', respectively;
when the mutation site to be identified is S371L, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 7: 5'-GTTGCTGATTATTCTGTCCTATATAATTTA-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 8: 5'-GTTGCTGATTATTCTGTCCTATATAAT TCC-3', the downstream primer is shown in SEQ ID NO. 9: 5'-GTCTGACTTCATCACCTCTAATTAC A-3', respectively;
when the mutation site to be identified is G446S, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 10: 5'-TGGAATTCTAACAATCTTGATTCTAAGGTTA-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 11: 5'-TGGAATTCTAACAATCTTGA TTCTAAGGTTG-3', the downstream primer is shown in SEQ ID NO. 12: 5'-GGAAACCATATGATT GTAAAGGAAAGTAAC-3', respectively;
step 2.2, designing a TaqMan probe: designing a TaqMan probe according to a general principle of TaqMan probe design and combining the sequence characteristics of the viral genome region limited by the upstream and downstream primers in the step 2.1, wherein two pairs of primers of each mutation site share one TaqMan probe;
when the mutation site to be identified is A67V, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 13: 5'-CATGTCTCTGGGACCAATGGTACTAAGAGG-3', respectively;
when the mutation site to be identified is N211I, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 14: 5'-CGTGATCTCCCTCAGGGTTTTTCGGC-3', respectively;
when the mutation site to be identified is S371L, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 15: 5'-CGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCC-3', respectively;
when the mutation site to be identified is G446S, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 16: 5'-CAGGCCGGTAGCACACCTTGTAATGGTGTT-3' are provided.
Step 3, performing qRT-PCR experiment based on ARMS;
and 4, interpretation of results:
step 4.1, judging that the mutation occurs in the detection sample when the occurrence time of the amplification curve of the mutation primer system is more than 2 cycles earlier than that of the non-mutation primer system;
and 4.2, judging that no variation occurs in the detection sample when the occurrence time of the amplification curve of the non-variation primer system is more than 2 cycles earlier than that of the variation primer system.
Further, the step 3 is based on ARMS to carry out qRT-PCR experiment, and comprises the following specific steps:
step 3.1, preparing a reaction system: each reaction system has the volume of 20 mu L, wherein the reaction system contains 10 mu L of 2X reaction liquid, 0.2 mu L of 50 mu M upstream primer, 0.2 mu L of 50 mu M downstream primer, 0.1 mu L of 50 mu M TaqMan probe, 7.5 mu L of sterile RNase-free water and 2 mu L of RNA template, the detection of each mutation site consists of two reaction systems, one contains a mutation upstream primer, the other contains a non-mutation downstream primer, and the other components are the same;
step 3.2, qRT-PCR reaction conditions: the following reaction procedure was performed on a fluorescent quantitative PCR instrument: at 95 ℃ for 3 minutes; 45 cycles of 95 deg.C, 30 seconds to 60 deg.C, 30 seconds.
Compared with the prior art, the invention has the following advantages:
1. the detection speed is improved, and the dependence on expensive instruments is reduced. By applying the method, the identification speed of the novel coronavirus varieties is shortened from 4-6 hours to 2 hours, and the method is independent of an expensive sequencer and can be completed only by a common fluorescent quantitative PCR instrument, so that the dependence on expensive instruments such as the sequencer is reduced, virus detection users can complete virus variety identification in the unit, the time for sample transportation and the like is saved, and uncontrollable factors are reduced to the maximum extent;
2. and the detection sensitivity is improved. The method is characterized in that the virus nucleic acid variation is detected based on a TaqMan probe method qRT-PCR, the detection sensitivity can reach 1-15 copies/microliter, the detection of the virus nucleic acid variation can be realized by the qRT-PCR having an amplification signal, and the influence of a weak amplification signal on the detection of the nucleic acid variation based on gene sequencing is avoided.
3. The detection cost is reduced, and the detection flux is improved. The whole experimental process of the technology only comprises two steps of virus nucleic acid extraction and qRT-PCR, the experimental steps are few, the required material and labor cost is greatly reduced, and the high flux of the sample can be conveniently realized through a 96-hole or 384-hole reaction plate.
4. The operation pollution probability is small, and the high-throughput operation is easy. The existing first-generation sequencing technology needs multiple steps of virus RNA extraction, RT-PCR amplification, PCR product purification, on-machine sequencing and the like for detecting virus variation, the batch operation is easy to introduce operation pollution, the method only has two steps of virus RNA extraction and qRT-PCR operation, the corresponding operation pollution reduction probability of the steps is also reduced, and fewer operation steps are easy to realize the detection of a large number of samples at one time.
Drawings
FIG. 1 is a graph showing the sensitivity results of the present invention for identifying the A67V mutation site;
FIG. 2 is a graph showing the results of the sensitivity of the present invention for identifying the N211I mutation site;
FIG. 3 is a graph showing the results of the sensitivity of the present invention for identifying the variation site S371L;
FIG. 4 is a graph showing the sensitivity results of the present invention for identifying the G446S mutation site.
Detailed Description
Example 1
A qRT-PCR method for identifying the branch of a novel coronavirus Omicron variant BA-1 comprises the following steps:
step 1, extracting new coronavirus RNA by using a Trizol method;
step 2.1, primer design: according to the general principle of primer design, combining nucleotide sequences near the variation sites of the novel coronavirus Indian variety, and designing two pairs of primers for each variation site, wherein the 3' end of an upstream primer is respectively matched with a variation point and a non-variation point, namely a variation upstream primer and a non-variation upstream primer, and the two pairs of upstream primers share a downstream primer;
when the mutation site to be identified is A67V, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 1: 5'-CCAATGTTACTTGGTTCCATGT-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 2: 5'-CCAATGTTACTTGGTTCCATGC-3', the downstream primer is shown in SEQ ID NO. 3: 5'-TGTTAGACTTCTCAGTGGAAGC-3', respectively;
when the variation site to be identified is N211I, the nucleotide sequence of the variation upstream primer is shown in SEQ ID NO. 4: 5'-AATATATTCTAAGCACACGCCTATTAT-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 5: 5'-AATATATTCTAAGCACACGCCTATTAA-3', the downstream primer is shown in SEQ ID NO. 6: 5'-GAGTCAAATAACTTCTATGTAAAGCA AG-3', respectively;
when the mutation site to be identified is S371L, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 7: 5'-GTTGCTGATTATTCTGTCCTATATAATTTA-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 8: 5'-GTTGCTGATTATTCTGTCCTATATAAT TCC-3', the downstream primer is shown in SEQ ID NO. 9: 5'-GTCTGACTTCATCACCTCTAATTAC A-3', respectively;
when the mutation site to be identified is G446S, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 10: 5'-TGGAATTCTAACAATCTTGATTCTAAGGTTA-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 11: 5'-TGGAATTCTAACAATCTTGAT TCTAAGGTTG-3', the downstream primer is shown in SEQ ID NO. 12: 5'-GGAAACCATATGATT GTAAAGGAAAGTAAC-3', respectively;
step 2.2, designing a TaqMan probe: designing a TaqMan probe according to a general principle of TaqMan probe design and combining the sequence characteristics of the viral genome region limited by the upstream and downstream primers in the step 2.1, wherein two pairs of primers of each mutation site share one TaqMan probe;
when the mutation site to be identified is A67V, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 13: 5'-CATGTCTCTGGGACCAATGGTACTAAGAGG-3', respectively;
when the mutation site to be identified is N211I, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 14: 5'-CGTGATCTCCCTCAGGGTTTTTCGGC-3', respectively;
when the mutation site to be identified is S371L, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 15: 5'-CGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCC-3', respectively;
when the mutation site to be identified is G446S, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 16: 5'-CAGGCCGGTAGCACACCTTGTAATGGTGTT-3' are provided.
And 3, carrying out qRT-PCR experiment based on ARMS, and specifically comprising the following steps:
step 3.1, preparing a reaction system: each reaction system has the volume of 20 mu L, wherein the reaction system contains 10 mu L of 2X reaction liquid, 0.2 mu L of 50 mu M upstream primer, 0.2 mu L of 50 mu M downstream primer, 0.1 mu L of 50 mu M TaqMan probe, 7.5 mu L of sterile RNase-free water and 2 mu L of RNA template, the detection of each mutation site consists of two reaction systems, one contains a mutation upstream primer, the other contains a non-mutation downstream primer, and the other components are the same;
step 3.2, qRT-PCR reaction conditions: the following reaction procedure was performed on a fluorescent quantitative PCR instrument: at 95 ℃ for 3 minutes; 45 cycles of 95 deg.C, 30 seconds to 60 deg.C, 30 seconds.
And 4, interpretation of results:
step 4.1, judging that the mutation occurs in the detection sample when the occurrence time of the amplification curve of the mutation primer system is more than 2 cycles earlier than that of the non-mutation primer system;
and 4.2, judging that no variation occurs in the detection sample when the occurrence time of the amplification curve of the non-variation primer system is more than 2 cycles earlier than that of the variation primer system.
Example 2
Sensitivity experiments were performed on the method of the invention (template dilution 10)6-10-1Copy/microliter), the results are as follows:
as shown in fig. 1, when the mutation site to be identified is a67V, the detection sensitivity is 4.55 copies/microliter; as shown in fig. 2, when the mutation site to be identified is N211I, the detection sensitivity is 6.82 copies/microliter; as shown in fig. 3, when the mutation site to be identified is S371L, the detection sensitivity is 1.20 copies/microliter; as shown in FIG. 4, when the mutation site to be identified was G446S, the detection sensitivity was 12.32 copies/μ l.
Example 3
The precision of the repeatability test evaluation method refers to the closeness degree between a series of single measured values obtained by repeatedly measuring the same sample for many times under certain conditions, is an index of random error magnitude of reaction, and is divided into batch repeatability tests, batch-to-batch repeatability tests (day-to-day and day-to-day repeatability tests), operator/instrument repeatability tests and the like.
The repeatability (precision) of the method is examined, the coefficient of variation is used for measurement, and the experimental results are shown in table 1.
TABLE 1 repeatability (precision) of the process
The calculation method of the variation coefficient comprises the following steps:
coefficient of variation (%) - (Ct standard deviation/Ct average) × 100%.
As can be seen from Table 1:
the experimental variation coefficient range in the A67V batch is 0.66-2.77%, and the experimental variation coefficient range between batches is 0.51-3.48%; the experimental variation coefficient range in the N211I batch is 0.53-2.45%, and the experimental variation coefficient range between batches is 61-2.33%; S371L, the experimental variation coefficient range in batch is 0.23% -2.48%, and the experimental variation coefficient range between batches is 0.48% -2.35%; the experimental variation coefficient range in the G446S batch is 0.39% -1.84%, and the experimental variation coefficient range between batches is 0.74% -2.82%;
the experimental variation coefficients in batches of each mutation detection primer of the method are all less than 3 percent, and the experimental variation coefficients in batches are all less than 4 percent. Therefore, the method has good repeatability (precision).
Example 3
The linearity of the linear and range (linear analysis) analysis method is the ability to obtain an experimental result proportional to the concentration of a test substance in a sample within a given range, i.e., the range of the concentration of the test substance linearly related to the experimental result is an important index of the detection performance of the reaction method.
We have examined the linear detection range of the method, and the experimental results are shown in Table 2. Wherein the slope is known, the system of the method is in the template 106-101The copy/microliter can obtain a detection signal which is in linear relation with the template amount, which shows that the method has a wide linear detection range.
TABLE 2 Linear detection Range of the method
Sequence listing
SEQ ID NO.1：5’-CCAATGTTACTTGGTTCCATGT-3’
SEQ ID NO.2：5’-CCAATGTTACTTGGTTCCATGC-3’
SEQ ID NO.3：5’-TGTTAGACTTCTCAGTGGAAGC-3’
SEQ ID NO.4：5’-AATATATTCTAAGCACACGCCTATTAT-3’
SEQ ID NO.5：5’-AATATATTCTAAGCACACGCCTATTAA-3’
SEQ ID NO.6：5’-GAGTCAAATAACTTCTATGTAAAGCAAG-3’
SEQ ID NO.7：5’-GTTGCTGATTATTCTGTCCTATATAATTTA-3’
SEQ ID NO.8：5’-GTTGCTGATTATTCTGTCCTATATAATTCC-3’
SEQ ID NO.9：5’-GTCTGACTTCATCACCTCTAATTACA-3’
SEQ ID NO.10：5’-TGGAATTCTAACAATCTTGATTCTAAGGTTA-3’
SEQ ID NO.11：5’-TGGAATTCTAACAATCTTGATTCTAAGGTTG-3’
SEQ ID NO.12：5’-GGAAACCATATGATTGTAAAGGAAAGTAAC-3’
SEQ ID NO.13：5’-CATGTCTCTGGGACCAATGGTACTAAGAGG-3’
SEQ ID NO.14：5’-CGTGATCTCCCTCAGGGTTTTTCGGC-3’
SEQ ID NO.15：5’-CGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCC-3’
SEQ ID NO.16：5’-CAGGCCGGTAGCACACCTTGTAATGGTGTT-3’
Sequence listing
<110> university of Shanxi
<120> qRT-PCR method for identifying BA-1 branch of novel coronavirus Omicron variant
<160> 16
<170> SIPOSequenceListing 1.0
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ccaatgttac ttggttccat gt 22
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ccaatgttac ttggttccat gc 22
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tgttagactt ctcagtggaa gc 22
<210> 4
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aatatattct aagcacacgc ctattat 27
<210> 5
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aatatattct aagcacacgc ctattaa 27
<210> 6
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gagtcaaata acttctatgt aaagcaag 28
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gttgctgatt attctgtcct atataattta 30
<210> 8
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gttgctgatt attctgtcct atataattcc 30
<210> 9
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gtctgacttc atcacctcta attaca 26
<210> 10
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tggaattcta acaatcttga ttctaaggtt a 31
<210> 11
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tggaattcta acaatcttga ttctaaggtt g 31
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ggaaaccata tgattgtaaa ggaaagtaac 30
<210> 13
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
catgtctctg ggaccaatgg tactaagagg 30
<210> 14
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cgtgatctcc ctcagggttt ttcggc 26
<210> 15
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cgcatcattt tccactttta agtgttatgg agtgtctcc 39
<210> 16
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
caggccggta gcacaccttg taatggtgtt 30
Claims (2)
1. A qRT-PCR method for identifying the branch of a novel coronavirus Omicron variant BA-1 is characterized by comprising the following steps:
step 1, extracting new coronavirus RNA by using a Trizol method;
step 2, designing a qRT-PCR primer probe based on ARMS:
step 2.1, primer design: according to the general principle of primer design, combining nucleotide sequences near the variation sites of the novel coronavirus Indian variety, and designing two pairs of primers for each variation site, wherein the 3' end of an upstream primer is respectively matched with a variation point and a non-variation point, namely a variation upstream primer and a non-variation upstream primer, and the two pairs of upstream primers share a downstream primer;
when the mutation site to be identified is A67V, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 1: 5'-CCAATGTTACTTGGTTCCATGT-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 2: 5'-CCAATGTTACTTGGTTCCATGC-3', the downstream primer is shown in SEQ ID NO. 3: 5'-TGTTAGACTTCTCAGTGGAAGC-3', respectively;
when the variation site to be identified is N211I, the nucleotide sequence of the variation upstream primer is shown in SEQ ID NO. 4: 5'-AATATATTCTAAGCACACGCCTATTAT-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 5: 5'-AATATATTCTAAGCACACGCCTATTAA-3', the downstream primer is shown in SEQ ID NO. 6: 5'-GAGTCAAATAACTTCTATGTAAAGCA AG-3', respectively;
when the mutation site to be identified is S371L, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 7: 5'-GTTGCTGATTATTCTGTCCTATATAATTTA-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 8: 5'-GTTGCTGATTATTCTGTCCTATATAAT TCC-3', the downstream primer is shown in SEQ ID NO. 9: 5'-GTCTGACTTCATCACCTCTAATTAC A-3', respectively;
when the mutation site to be identified is G446S, the nucleotide sequence of the mutation upstream primer is shown in SEQ ID NO. 10: 5'-TGGAATTCTAACAATCTTGATTCTAAGGTTA-3', the nucleotide sequence of the non-variant upstream primer is shown in SEQ ID NO. 11: 5'-TGGAATTCTAACAATCTTGA TTCTAAGGTTG-3', the downstream primer is shown in SEQ ID NO. 12: 5'-GGAAACCATATGAT TGTAAAGGAAAGTAAC-3';
step 2.2, designing a TaqMan probe: designing a TaqMan probe according to a general principle of TaqMan probe design and combining the sequence characteristics of the viral genome region limited by the upstream and downstream primers in the step 2.1, wherein two pairs of primers of each mutation site share one TaqMan probe;
when the mutation site to be identified is A67V, the nucleotide sequence of the TaqMan probe is shown as SEQ ID NO. 13: 5'-CATGTCTCTGGGACCAATGGTACTAAGAGG-3', respectively;
when the mutation site to be identified is N211I, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 14: 5'-CGTGATCTCCCTCAGGGTTTTTCGGC-3', respectively;
when the mutation site to be identified is S371L, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 15: 5'-CGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCC-3', respectively;
when the mutation site to be identified is G446S, the nucleotide sequence of the TaqMan probe is shown in SEQ ID NO. 16: 5'-CAGGCCGGTAGCACACCTTGTAATGGTGTT-3' are provided.
Step 3, performing qRT-PCR experiment based on ARMS;
and 4, interpretation of results:
step 4.1, judging that the mutation occurs in the detection sample when the occurrence time of the amplification curve of the mutation primer system is more than 2 cycles earlier than that of the non-mutation primer system;
and 4.2, judging that the mutation does not occur in the detection sample when the occurrence time of the amplification curve of the non-mutation primer system is more than 2 cycles earlier than that of the mutation primer system.
2. The qRT-PCR method for identifying the branch BA-1 of the novel coronavirus Omicron variant according to claim 1, wherein the step 3 is carried out qRT-PCR experiment based on ARMS, and comprises the following specific steps:
step 3.1, preparing a reaction system: each reaction system has the volume of 20 mu L, wherein the reaction system contains 10 mu L of 2X reaction liquid, 0.2 mu L of 50 mu M upstream primer, 0.2 mu L of 50 mu M downstream primer, 0.1 mu L of 50 mu M TaqMan probe, 7.5 mu L of sterile RNase-free water and 2 mu L of RNA template, the detection of each mutation site consists of two reaction systems, one contains a mutation upstream primer, the other contains a non-mutation downstream primer, and the other components are the same;
step 3.2, qRT-PCR reaction conditions: the following reaction procedure was performed on a fluorescent quantitative PCR instrument: at 95 ℃ for 3 minutes; 45 cycles of 95 deg.C, 30 seconds-60 deg.C, 30 seconds.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210187453.4A CN114480742A (en) | 2022-02-28 | 2022-02-28 | qRT-PCR method for identifying BA-1 branch of novel coronavirus Omicron variant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210187453.4A CN114480742A (en) | 2022-02-28 | 2022-02-28 | qRT-PCR method for identifying BA-1 branch of novel coronavirus Omicron variant |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114480742A true CN114480742A (en) | 2022-05-13 |
Family
ID=81483784
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210187453.4A Pending CN114480742A (en) | 2022-02-28 | 2022-02-28 | qRT-PCR method for identifying BA-1 branch of novel coronavirus Omicron variant |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114480742A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506067A (en) * | 2014-10-20 | 2016-04-20 | 江苏所罗门兄弟医学科技有限公司 | AMRMS-qPCR detection kit and detection method for NOSLAP genotyping |
| CN113249525A (en) * | 2021-06-25 | 2021-08-13 | 山西大学 | qRT-PCR method for identifying novel coronavirus Indian variety |
| WO2021175298A1 (en) * | 2020-03-05 | 2021-09-10 | 浙江东方基因生物制品股份有限公司 | Novel coronavirus detection reagent and detection method |
| WO2021189546A1 (en) * | 2020-03-27 | 2021-09-30 | 中国科学院合肥物质科学研究院 | Kit for detecting sars-cov-2 coronavirus, and specific primer and probe thereof |
| CN114085928A (en) * | 2022-01-19 | 2022-02-25 | 广东和信健康科技有限公司 | Rapid detection system for typing of novel coronavirus Omicron mutant strain |
-
2022
- 2022-02-28 CN CN202210187453.4A patent/CN114480742A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506067A (en) * | 2014-10-20 | 2016-04-20 | 江苏所罗门兄弟医学科技有限公司 | AMRMS-qPCR detection kit and detection method for NOSLAP genotyping |
| WO2021175298A1 (en) * | 2020-03-05 | 2021-09-10 | 浙江东方基因生物制品股份有限公司 | Novel coronavirus detection reagent and detection method |
| WO2021189546A1 (en) * | 2020-03-27 | 2021-09-30 | 中国科学院合肥物质科学研究院 | Kit for detecting sars-cov-2 coronavirus, and specific primer and probe thereof |
| CN113249525A (en) * | 2021-06-25 | 2021-08-13 | 山西大学 | qRT-PCR method for identifying novel coronavirus Indian variety |
| CN114085928A (en) * | 2022-01-19 | 2022-02-25 | 广东和信健康科技有限公司 | Rapid detection system for typing of novel coronavirus Omicron mutant strain |
Non-Patent Citations (2)
| Title |
|---|
| SURESH KUMAR等: "Omicron (BA.1) and Sub-Variants (BA.1, BA.2 and BA.3) of SARS-CoV-2 Spike Infectivity and Pathogenicity: A Comparative Sequence and Structural-based Computational Assessment", BIORXIV, pages 1 - 20 * |
| 徐昌平;卢亦愚;严菊英;冯燕;高筱萍;: "TaqMan荧光定量RT-PCR快速检测西尼罗病毒的研究", 中国媒介生物学及控制杂志, no. 02, pages 53 - 55 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113249525A (en) | qRT-PCR method for identifying novel coronavirus Indian variety | |
| Reid et al. | Authentication of human cell lines by STR DNA profiling analysis | |
| EP1131466B1 (en) | Method for the quantitative detection of nucleic acids | |
| WO2021238087A1 (en) | Novel coronavirus rapid detection kit based on thermal convection pcr | |
| CN113652505B (en) | Method and kit for detecting novel coronavirus and VOC-202012/01 mutant strain thereof | |
| WO1993002215A1 (en) | Quantitative nucleic acid amplification | |
| CN115852058A (en) | Primer and probe combination for detecting T cell lymphotropic virus of monkey and application thereof | |
| CN114480742A (en) | qRT-PCR method for identifying BA-1 branch of novel coronavirus Omicron variant | |
| CN116716415A (en) | Primer probe for quantitative detection of DNA residual content of insect cells, method and application thereof | |
| CN114657285A (en) | qRT-PCR method for identifying BA-2 branch of novel coronavirus Omicron variant | |
| CN113481324B (en) | Method and kit for detecting novel coronavirus and D614G mutant thereof | |
| CN114164303A (en) | qRT-PCR method for identifying novel coronavirus Gamma variant strain | |
| CN114277194A (en) | qRT-PCR method for identifying novel coronavirus Lambda variant | |
| CN108842001A (en) | For detecting the primer and probe, PCR kit for fluorescence quantitative and methods and applications of pig Delta coronavirus | |
| CN114438263A (en) | Fluorescent quantitative PCR (polymerase chain reaction) primer group and kit for detecting European eel circovirus | |
| CN106755392B (en) | qPCR (quantitative polymerase chain reaction) method for rapidly and quantitatively detecting coelomacter in algae culture | |
| CN111206117A (en) | Kit for detecting human immunodeficiency virus | |
| CN114317822A (en) | Multiplex fluorescence quantitative PCR method for detecting new coronavirus nucleic acid | |
| CN116334189B (en) | Internal standard system for monitoring qPCR reaction interference and application thereof | |
| CN106755393B (en) | qPCR (quantitative polymerase chain reaction) method for rapidly and quantitatively detecting Brachionus brachialis in algae culture | |
| CN113881808B (en) | Physical titer detection kit and detection method for viral vector | |
| CN111424074B (en) | Nucleic acid detection kit for quantifying trace residue of mouse source tissue | |
| CN114561496B (en) | Primer and probe for identifying novel coronavirus Delta strain and application of primer and probe | |
| CN114150088B (en) | New coronavirus gene single base mutation detection method and application thereof | |
| CN118755860A (en) | QPCR method for detecting host cell DNA residue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |