CN112708698A - Primer group for determining CVB3 virus whole gene sequence - Google Patents
Primer group for determining CVB3 virus whole gene sequence Download PDFInfo
- Publication number
- CN112708698A CN112708698A CN202011568704.0A CN202011568704A CN112708698A CN 112708698 A CN112708698 A CN 112708698A CN 202011568704 A CN202011568704 A CN 202011568704A CN 112708698 A CN112708698 A CN 112708698A
- Authority
- CN
- China
- Prior art keywords
- cvb3
- seq
- primer
- gene sequence
- virus
- 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
- 241000709675 Coxsackievirus B3 Species 0.000 title claims abstract description 52
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 50
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- 238000012163 sequencing technique Methods 0.000 claims abstract description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 3
- 108050009160 DNA polymerase 1 Proteins 0.000 claims description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 2
- 239000002299 complementary DNA Substances 0.000 claims description 2
- 239000012634 fragment Substances 0.000 abstract description 14
- 238000000034 method Methods 0.000 abstract description 10
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 230000003321 amplification Effects 0.000 abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 238000011841 epidemiological investigation Methods 0.000 abstract description 2
- 239000002773 nucleotide Substances 0.000 description 28
- 125000003729 nucleotide group Chemical group 0.000 description 28
- 241000700605 Viruses Species 0.000 description 12
- 208000015181 infectious disease Diseases 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 238000011144 upstream manufacturing Methods 0.000 description 8
- 208000020061 Hand, Foot and Mouth Disease Diseases 0.000 description 7
- 208000025713 Hand-foot-and-mouth disease Diseases 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 238000001179 sorption measurement Methods 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 241000709687 Coxsackievirus Species 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 6
- 241000709661 Enterovirus Species 0.000 description 6
- 239000003480 eluent Substances 0.000 description 6
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 2
- 241000146367 Coxsackievirus A10 Species 0.000 description 2
- 241001429382 Coxsackievirus A16 Species 0.000 description 2
- 241001669084 Coxsackievirus A6 Species 0.000 description 2
- 241001529459 Enterovirus A71 Species 0.000 description 2
- 241001207270 Human enterovirus Species 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 206010014599 encephalitis Diseases 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 201000011475 meningoencephalitis Diseases 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 206010047470 viral myocarditis Diseases 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- 206010051093 Cardiopulmonary failure Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 208000028399 Critical Illness Diseases 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000001738 Nervous System Trauma Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- 208000033952 Paralysis flaccid Diseases 0.000 description 1
- 206010034960 Photophobia Diseases 0.000 description 1
- 241000709664 Picornaviridae Species 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 101150024766 VP1 gene Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 208000028331 flaccid paralysis Diseases 0.000 description 1
- 208000018685 gastrointestinal system disease Diseases 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 208000028412 nervous system injury Diseases 0.000 description 1
- 201000005737 orchitis Diseases 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a primer group for determining CVB3 virus whole gene sequence, comprising three pairs of primers, wherein the nucleotide sequences are respectively shown as SEQ ID NO: 1-2, SEQ ID NO: 3-4 and SEQ ID NO: 5 to 6. The invention utilizes 3 pairs of primers with strong specificity, high sensitivity and good stability to carry out sequence amplification on 11 fragment genes (including VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3B, 3C and 3D fragments) of the CVB3 virus through 3 conventional PCR reactions, and then carries out fragment sequencing to efficiently determine the CVB3 virus whole gene. The method for detecting the CVB3 by using the 3 pairs of specific primers is simple to operate, short in time consumption, strong in specificity, high in sensitivity and suitable for identification of the CVB3 virus, epidemiological investigation and research and the like.
Description
Technical Field
The invention relates to the technical field of CVB3 virus detection, in particular to a primer group for determining a CVB3 virus whole gene sequence.
Background
Hand-foot-and-mouth disease (HFMD) is a type of epidemic infectious disease caused by Human Enterovirus (HEV) infection, mostly occurs in infants under 5 years old, and can cause serious central nervous system injury, cardiopulmonary failure and other symptoms, and HFMD is a third-class infectious disease legally reported in China in 5 months 2008. In recent years, the cases caused by HFMD are in a high-incidence state, seriously endanger the health and life safety of infants, and bring heavy diseases and economic burden to families. However, currently, our country mainly focuses on enterovirus type 71 (enterovirus a71, EVA71), coxsackievirus type a16 (coxsackievirus a16, CVA16), coxsackievirus type a10 (coxsackievirus a10, CVA10) and coxsackievirus type a6 (coxsackievirus a6, CVA6), and the identification of the types of other enteroviruses is not enough, but the proportion of other enteroviruses is increased in recent years. In addition, the etiologic spectrum of HFMD contributes to diversification and complexity.
(coxsackieviruses group B, CVB) belong to the genus Enterovirus of the family picornaviridae, for a total of 6 family members, CVB 1-6. It is a single plus strand full-length RNA virus with the size of about 30nm, nucleocapsid icosahedral stereo symmetry and no envelope, about 7.5 kb. The genome has only one open reading frame, which can encode 4 structural genes (VP1-VP4) and 7 non-structural genes (2A-2C and 3A-3D), and 2 non-coding regions (5 'UTR and 3' UTR). Although coxsackie virus belongs to enteroviruses and its mode of transmission through the feces, the virus does not cause gastrointestinal disease. Many coxsackie group B viruses infect humans with mild symptoms, usually only produce cold-like symptoms, but can cause severe diseases such as viral myocarditis, pneumonia, hepatitis, pancreatitis, orchitis and the like in immunocompromised patients and in some elderly and young children. Coxsackie B3 virus (CVB3) has been reported by the united states diagnostic laboratories to be within the 15 most common enterovirus list of the CDC, with a high incidence of infection, particularly in young children, capable of causing infections of the liver and lungs of newborn infants, being the leading causative agent of viral myocarditis infection in newborn infants, with a few critically ill patients at risk of death. Epidemiological studies, RNA detection, and serological analyses have demonstrated that CVB3 infection is linked to the presence of Type 1diabetes (T1D). In addition, CVB3 can invade the central nervous system, and can cause aseptic meningoencephalitis and acute flaccid paralysis, and clinical symptoms such as fever, meningoencephalia, headache, photophobia and the like appear in the onset stage. Clinical diagnosis is based on the clinical manifestations of the patient and is aided by the evaluation of cerebrospinal fluid laboratory tests, electroencephalograms, imaging CT and MRI. In the last 20 years, large-scale enterovirus outbreak infection has been reported worldwide, causing neurological disorders and meningoencephalitis, and CVB3 is also the main source of infection.
At present, the clinical diagnosis CVB3 takes detected virus nucleic acid as one of the confirmed diagnosis indexes, but the current methods related to the whole gene sequence typing of the CVB3 virus have few reports, and in order to enhance the monitoring and prevention of HFMD caused by CVB3, the complete gene sequence information of the CVB3 is clear, and the method plays a great role in researching the aspects of virulence, infectivity and the like of different virus strains. The invention provides the possibility of the development, not only lays a foundation for epidemiological analysis, pathogenic mechanism and the like of the hand-foot-and-mouth disease in the future, but also provides a new idea for detecting virus infection for clinical and scientific research.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a primer group for determining the whole gene sequence of CVB3 virus.
The first purpose of the invention is to provide a primer group for determining the whole gene sequence of CVB3 virus.
The second purpose of the invention is to provide the application of one or more pairs of the primer sets in the preparation of a complete gene sequence determination kit of the CVB3 virus.
The third purpose of the invention is to provide a complete gene sequence determination kit of CVB3 virus.
In order to achieve the purpose, the invention is realized by the following scheme:
the invention claims a primer group for determining a CVB3 virus complete gene sequence, which is characterized by comprising three pairs of primers, wherein the nucleotide sequences of the primers are respectively shown as SEQ ID NO: 1-2, SEQ ID NO: 3-4 and SEQ ID NO: 5 to 6.
1, the nucleotide sequence of a primer used for detecting and identifying CVB3 virus 5' -UTR gene segments, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, as shown in the figure:
the nucleotide sequence of the upstream primer 5' UTR-F is shown as SEQ ID NO: as shown at 1 (ACAGCCTGTGGGTTGYHCCCA), and,
the nucleotide sequence of the downstream primer 5' UTR-R is shown as SEQ ID NO: 2 (AAGTAGTCGGTTCCGCTGCAGAGTT);
and 2, the nucleotide sequence of a primer used for detecting and identifying CVB3 virus 5' -UTR to 2A gene segments is shown as SEQ ID NO: 3, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 4, and (2) is as follows:
the nucleotide sequence of the upstream primer 5' UTR-2A-F is shown as SEQ ID NO: as shown at (GGAACCGACTACTTTGGGTGTCCGTGTTTC) in figure 3,
the nucleotide sequence of the downstream primer 5' UTR-2A-R is shown as SEQ ID NO: 4 (CTGYTCCATKGCRTCATCTTCCARCC);
the 3 rd counterpart is used for detecting and identifying the primer nucleotide sequence of 2A to 3D gene segments of CVB3 virus, and the nucleotide sequence of an upstream primer is shown as SEQ ID NO: 5, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 6, showing:
the nucleotide sequence of the upstream primer 2A-3D-F is shown as SEQ ID NO: as shown in figure 5 (GGRCARCAATCDGGRGCARYRTAYGT),
the nucleotide sequence of the downstream primer 2A-3D-R is shown as SEQ ID NO: shown at 6 (AAAGGAGTCCAACCACTTCCTGC).
3 pairs of specific primers are divided into 3 groups for detecting CVB3 virus whole gene coding sequences, and CVB3 virus whole genes comprise eleven segments of VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3B, 3C and 3D;
the application of one or more pairs of primer sets in the preparation of a complete gene sequence determination kit of the CVB3 virus also belongs to the protection scope of the invention.
The invention also claims a complete gene sequence determination kit of the CVB3 virus, which contains one or more pairs of the primer groups.
Preferably, the primer set is contained.
Preferably, reagents for the PCR reaction are also contained.
Preferably, the reagents for the PCR reaction are dNTP Mix, Phanta Max Super-Fidelity DNA Polymerase, and 2 × Phanta Max Buffer.
Preferably, the procedure for the PCR reaction is: 3min at 95 ℃; circulating for 30 times at 95 ℃ for 15s, 60 ℃ for 15s and 72 ℃ for 60 s; 72 ℃ for 5 min.
Preferably, the PCR reaction system: 2X Phanta Max Buffer 25. mu.l; 10 μ M of the forward primer 2 μ l; 10 μ M downstream primer 2 μ l; 10 μ M each dNTP Mix 1 μ l; phanta Max Super-Fidelity DNA Polymerase 1. mu.l; 1 μ l of cDNA; sterile ddH2O 18μl。
Compared with the prior art, the invention has the following beneficial effects:
the advantages are as follows:
1. the operation is simple, the result is stable, 3 PCR reaction processes are all carried out under the same condition, and the efficiency is high.
2. The sensitivity is high, and 3 PCR reactions can be efficiently carried out.
3. The specificity is strong, and 3 PCR reaction products are single electrophoresis bands.
The invention utilizes 3 pairs of primers with strong specificity, high sensitivity and good stability to carry out sequence amplification on 11 fragment genes (including VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3B, 3C and 3D fragments) of the CVB3 virus through 3 conventional PCR reactions, and then carries out fragment sequencing to efficiently determine the CVB3 virus whole gene. The method for detecting the CVB3 by using the 3 pairs of specific primers is simple to operate, short in time consumption, strong in specificity, high in sensitivity and suitable for identification of the CVB3 virus, epidemiological investigation and research and the like.
Drawings
FIG. 1 is an agarose gel electrophoresis image of whole gene segmented PCR of a CVB3 strain to be tested (accession number CVB3-5341) using the primers of the present invention.
FIG. 2 comparison of VP1 fragment homology of CVB3-5341 virus strain with other CVB3 virus strains that have submitted GenBank full gene sequences.
FIG. 3 comparison of genomic homology of CVB3-5341 strain with other CVB3 strains that have submitted a GenBank complete gene sequence.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
EXAMPLE 1 design of primers and probes
First, experiment method
By respectively carrying out alignment analysis on the gene sequences of all known CVB3 virus strains in a GenBank database, highly conserved segments are selected, and primers are designed according to the following principle:
(1) the length of the primer is about 20-23 nt, and the GC content is 45-55%.
(2) For the same template segment, primers are designed to enable each site to be covered by 2-3 PCR amplification segments, so that the influence of point mutation on the result interpretation caused by PCR reaction is avoided.
The information of the above virus-specific genes was analyzed, the elimination of inter/intra primer dimers was performed by the sequence analysis software DNASTAR, and the homology of the specificity of the primers to the similar pathogens was verified by BLAST, and primers for detection of the above pathogens were designed.
Second, experimental results
3 pairs of primers are designed, and the nucleotide sequences are shown as SEQ ID NO: 1 to 6.
The nucleotide sequence of the primer 5' UTR-F is shown as SEQ ID NO: 1 (ACAGCCTGTGGGTTGYHCCCA);
the nucleotide sequence of the primer 5' UTR-R is shown as SEQ ID NO: 2 (AAGTAGTCGGTTCCGCTGCAGAGTT);
the nucleotide sequence of the primer 5' UTR-2A-F is shown as SEQ ID NO: shown at 3 (GGAACCGACTACTTTGGGTGTCCGTGTTTC);
the nucleotide sequence of the primer 5' UTR-2A-R is shown as SEQ ID NO: 4 (CTGYTCCATKGCRTCATCTTCCARCC);
the nucleotide sequence of the primer 2A-3D-F is shown as SEQ ID NO: shown at 5 (GGRCARCAATCDGGRGCARYRTAYGT);
the nucleotide sequence of the primer 2A-3D-R is shown as SEQ ID NO: shown at 6 (AAAGGAGTCCAACCACTTCCTGC).
3 pairs of specific primers are divided into 3 groups for detecting CVB3 virus whole gene coding sequences, and CVB3 virus whole genes comprise eleven segments of VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3B, 3C and 3D;
example 2 Whole Gene sequence determination kit for CVB3 Virus
A, make up
The nucleotide sequence is shown as SEQ ID NO: 1 to 6, in a primer set,
second, use method
1. Extraction of sample RNA
Viral RNA was extracted from throat swab specimens using the QIAamp MinElute Virus Spin kit (Qiagen, Chatsworth, CA, catalog #74104) according to the following protocol:
(1) adding 25 mul Qiagen Protease into a new 1.5ml centrifuge tube, adding the sample to be tested, and mixing uniformly;
(2) adding 200 μ l of adsorption solution into each tube, mixing, and heating at 56 deg.C for 15 min;
(3) after heating, 250 mul of absolute ethyl alcohol is added into each tube, and after uniform mixing, the mixture is placed for 5min at room temperature.
(4) Adding the mixed solution into an adsorption column, centrifuging and removing waste liquid;
(5) adding 500 μ l of eluent 1 into the adsorption column, centrifuging, and discarding waste liquid;
(6) adding 500 μ l of eluent 2 into the adsorption column, centrifuging, and discarding waste liquid;
(7) adding 500 μ l of anhydrous ethanol into the adsorption column, centrifuging, and removing waste liquid;
(8) the adsorption was placed on another clean collection tube, left empty 14000rpm for 3 min.
(9) The column sample was eluted with 30 μ and collected.
2. RNA purity and concentration determination
Mu.l of the RNA solution was taken, and absorbance values (A) A at wavelengths of 260nm and 280nm were measured and calculated using a Nano Drop1000 nucleic acid protein quantifier260And A280A is more than or equal to 1.8 and RNA concentration260/A280The purity is less than or equal to 2.0, which indicates that the purity meets the requirement. Based on the measured concentration, all RNAs were diluted with RNase-free water to a final concentration of 1. mu.g/. mu.l.
2. Carrying out a reverse transcription reaction
(1) System configuration: the Vazyme company was used
II Q Select RT Supermix for qPCR reverse transcription reaction, reaction components are configured as follows:
(2) placing the reaction tube added with the reaction system in a PCR instrument for RT-PCR, wherein the reaction procedure is as follows:
4. PCR reaction system and conditions
The individual reaction systems are as follows:
the reaction procedure of the PCR is as follows:
the amplification products were detected by electrophoresis on a 1.5% agarose gel containing ethidium bromide at a final concentration of 0.5. mu.g/ml. And (4) sequencing the amplified product if the amplified product has a single specific band (otherwise, sequencing the amplified product by adjusting the PCR reaction condition until the electrophoresis detects the single specific band).
After sequencing was completed, sequencing results of the same gene fragments were aligned and integrated into a complete sequence using the bioanalytical software DNASTAR.
5. Gel recovery purification of PCR products
Gel recovery and purification were performed with a QIAquick Gel Extraction kit (Qiagen, Chatsworth, Calif.), and the specific experimental steps were arranged as follows according to the kit instructions:
the Buffer QG, the experiment Buffer reagent and the Collection Tube, Spin Column consumables referred to below are all provided in the kit.
(1) The agarose gel containing the PCR product bands was cut under uv light, weighed and the gel block volume calculated (1 μ g to 1 μ l);
(2) adding 3 buffers QG with gel volume, mixing uniformly, and heating at 50 ℃ to melt the gel blocks;
(3) transferring the melted mixed solution to Spin Column on Collection Tube provided by the kit, centrifuging at 12000rpm for 1min, and removing the filtrate;
(4) adding 750 ul Buffer PE into Spin Column, centrifuging at 12000rpm for 30 s;
(5) discarding the filtrate, and centrifuging at 12000rpm for 30s again;
(6) the Spin Column was placed in a new 1.5ml centrifuge tube, 50. mu.l of an Elution Buffer preheated at 65 ℃ was added to the center of the Spin Column membrane, and after standing at room temperature for 1min, it was centrifuged at 12000rpm for 1min, and the eluted PCR product was stored at-20 ℃.
6. Analysis of sequencing results
Sequencing results of three sets of amplified gene fragments were aligned and integrated into a complete sequence using the bioanalytical software DNASTAR. And (3) performing sequence similarity analysis on the sequenced virus strain, and if the sequence similarity and homology are high, indicating that the tested virus strain is CVB 3.
EXAMPLE 2 detection of Positive samples
First, experiment method
The kit of example 2 was used to detect the coxsackie virus CVB3 strain, which was provided by the laboratory on stock (using inactivated virus as the sample to be tested).
Second, experimental results
The CVB3-5341 strain was sequenced from the viral genome using the kit of example 2, and the PCR products were subjected to agarose gel electrophoresis as shown in FIG. 1.
Sequencing results of the same gene fragments were aligned and integrated into the complete sequence using the bioanalytical software DNASTAR. VP1 sequence and gene full-length similarity analysis were performed on the sequenced virus strain CVB 3. The results showed that compared with six CVB3 strains (CVB3-Nancy, CVB3-USA2018-23091, CVB3-XZ2011025, CVB3-Fuyang19, CVB3-MCH) already filed in GenBank, the VP1 gene fragment of CVB3 has more than 79% of nucleic acid homology (fig. 2), especially more than 90% of nucleic acid homology with two domestic CVB3 strains (CVB3-XZ2011025 and CVB3-Fuyang19), and the eleven gene fragments (VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3B, 3C and 3D) of CVB3 have more than 79% of nucleic acid homology (fig. 3). Sequencing results and sequence similarity analysis indicated that the viral strain identified in the present invention was CVB 3.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
<110> Zhongshan university
<120> a primer set for determining the whole gene sequence of CVB3 virus
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
acagcctgtg ggttgyhccc a 21
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aagtagtcgg ttccgctgca gagtt 25
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggaaccgact actttgggtg tccgtgtttc 30
<210> 4
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ctgytccatk gcrtcatctt ccarcc 26
<210> 5
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggrcarcaat cdggrgcary rtaygt 26
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aaaggagtcc aaccacttcc tgc 23
Claims (8)
1. A primer group for determining CVB3 virus whole gene sequence is characterized by comprising three pairs of primers, wherein the nucleotide sequences of the primers are respectively shown as SEQ ID NO: 1-2, SEQ ID NO: 3-4 and SEQ ID NO: 5 to 6.
2. Use of one or more pairs of the primer set of claim 1 in the preparation of a complete gene sequence determination kit for the CVB3 virus.
3. A kit for sequencing the entire gene of CVB3, comprising one or more pairs of the primer set of claim 1.
4. The kit for determining the whole gene sequence according to claim 3, comprising the primer set according to claim 1.
5. The kit for determining the whole gene sequence according to claim 4, further comprising a reagent for PCR reaction.
6. The kit for determining the whole gene sequence according to claim 5, wherein the reagents for PCR reaction are dNTP Mix, Phanta Max Super-Fidelity DNA Polymerase, and 2 x Phanta Max Buffer.
7. The kit for determining the whole gene sequence according to claim 3, wherein the PCR reaction is performed by the following steps: 3min at 95 ℃; circulating for 30 times at 95 ℃ for 15s, 60 ℃ for 15s and 72 ℃ for 60 s; 72 ℃ for 5 min.
8. The whole gene sequence determination kit according to claim 6, wherein the PCR reaction system comprises: 2X Phanta Max Buffer 25. mu.l; 10 μ M of the forward primer 2 μ l; 10 μ M downstream primer 2 μ l; 10 μ M each dNTP Mix 1 μ l; phanta Max Super-Fidelity DNA Polymerase 1. mu.l; 1 μ l of cDNA; sterieddH2O 18μl。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011568704.0A CN112708698A (en) | 2020-12-25 | 2020-12-25 | Primer group for determining CVB3 virus whole gene sequence |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011568704.0A CN112708698A (en) | 2020-12-25 | 2020-12-25 | Primer group for determining CVB3 virus whole gene sequence |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112708698A true CN112708698A (en) | 2021-04-27 |
Family
ID=75546767
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011568704.0A Pending CN112708698A (en) | 2020-12-25 | 2020-12-25 | Primer group for determining CVB3 virus whole gene sequence |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112708698A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000058524A2 (en) * | 1999-03-31 | 2000-10-05 | The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services | Typing of human enteroviruses |
| CN105525039A (en) * | 2016-01-28 | 2016-04-27 | 中国医学科学院医学生物学研究所 | Amplification method for whole genome aiming at different enterovirus serotypes |
| US20190099460A1 (en) * | 2017-09-29 | 2019-04-04 | Technische Universität Berlin | Method for Treating Cancer with a Coxsackievirus B3 (CVB3) Variant |
| CN110387438A (en) * | 2019-07-08 | 2019-10-29 | 广东省公共卫生研究院 | Multi-primers, kit and method for enterovirus high-flux sequence |
| CN111534637A (en) * | 2020-04-27 | 2020-08-14 | 江苏派森杰生物科技有限公司 | Universal primer, probe and kit for enterovirus nucleic acid detection |
-
2020
- 2020-12-25 CN CN202011568704.0A patent/CN112708698A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000058524A2 (en) * | 1999-03-31 | 2000-10-05 | The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services | Typing of human enteroviruses |
| CN105525039A (en) * | 2016-01-28 | 2016-04-27 | 中国医学科学院医学生物学研究所 | Amplification method for whole genome aiming at different enterovirus serotypes |
| US20190099460A1 (en) * | 2017-09-29 | 2019-04-04 | Technische Universität Berlin | Method for Treating Cancer with a Coxsackievirus B3 (CVB3) Variant |
| CN110387438A (en) * | 2019-07-08 | 2019-10-29 | 广东省公共卫生研究院 | Multi-primers, kit and method for enterovirus high-flux sequence |
| CN111534637A (en) * | 2020-04-27 | 2020-08-14 | 江苏派森杰生物科技有限公司 | Universal primer, probe and kit for enterovirus nucleic acid detection |
Non-Patent Citations (3)
| Title |
|---|
| HONGJIE LI ET AL.: "Complete genome sequence of a new recombinant echovirus 25 strain isolated from a neonatal patient with hand, foot, and mouth disease complicated by encephalitis in Beijing, China", 《VIRUS GENES》 * |
| 卞莲莲 等: "江苏省2012年柯萨奇病毒Ｂ组3型分离株全基因序列分析", 《中国病毒病杂志》 * |
| 韩振志 等: "两株西藏ＣＶ－Ｂ３的全基因组特征及溯源分析", 《病毒学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mirand et al. | Prospective identification of enteroviruses involved in meningitis in 2006 through direct genotyping in cerebrospinal fluid | |
| Nasri et al. | Typing of human enterovirus by partial sequencing of VP2 | |
| US11834723B2 (en) | Methods and compositions for detection of enterovirus D68 | |
| CN111534633B (en) | Novel coronavirus (2019-nCOV) detection kit and method | |
| Tan et al. | Specific detection of enterovirus 71 directly from clinical specimens using real-time RT-PCR hybridization probe assay | |
| CN113249522A (en) | Method for detecting SARS-CoV-2variant strain nucleic acid and its application | |
| Wang et al. | Rapid detection of hand, foot and mouth disease enterovirus genotypes by multiplex PCR | |
| CN112063753A (en) | Locked nucleic acid modified primer pair, method and kit for detecting African swine fever virus | |
| CN113122655A (en) | TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method for African swine fever virus EP402R gene | |
| Wang et al. | Rapid differentiation of PEDV wild-type strains and classical attenuated vaccine strains by fluorescent probe-based reverse transcription recombinase polymerase amplification assay | |
| CN112593011A (en) | Primer and probe for detecting coxsackie virus B group | |
| CN112280899A (en) | Porcine astrovirus type 2 TaqMan fluorescent quantitative PCR kit and application thereof | |
| Liu et al. | Coxsackievirus B: the important agent of hand, foot, and mouth disease | |
| Siafakas et al. | Molecular detection and identification of enteroviruses in children admitted to a university hospital in Greece | |
| CN112501356A (en) | Probe primer group, kit and detection method for rapidly identifying and detecting mink coronavirus and novel coronavirus | |
| CN112708698A (en) | Primer group for determining CVB3 virus whole gene sequence | |
| CN114438265B (en) | Nucleic acid composition, kit and detection method for simultaneously detecting porcine delta coronavirus, reovirus and porcine kokumi virus | |
| CN112626278B (en) | Primer and probe for identifying canine distemper virus wild strain and vaccine strain and application | |
| CN112626271A (en) | Primer composition for typing and/or detecting enteroviruses EV-A and EV-B | |
| CN109777888A (en) | Primer combination that is a kind of while detecting a variety of A group Human enterovirus virus and its application | |
| Castro et al. | Echovirus 30 associated with cases of aseptic meningitis in state of Pará, Northern Brazil | |
| Shaukat et al. | Characterization of non-polio enterovirus isolates from acute flaccid paralysis children in Pakistan reflects a new genotype of EV-107 | |
| Yoo et al. | Optimization of RT-PCR methods for enterovirus detection in groundwater | |
| Osazuwa et al. | Genetic diversity of norovirus among children under 5 years in the South-South region of Nigeria | |
| CN116144836B (en) | Quadruple fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer set for PRRSV (porcine reproductive and respiratory syndrome virus) and detection method |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210427 |