CN116716437A - Tick-derived vitex virus genome amplification primer group, amplification method and application - Google Patents
Tick-derived vitex virus genome amplification primer group, amplification method and application Download PDFInfo
- Publication number
- CN116716437A CN116716437A CN202310651946.3A CN202310651946A CN116716437A CN 116716437 A CN116716437 A CN 116716437A CN 202310651946 A CN202310651946 A CN 202310651946A CN 116716437 A CN116716437 A CN 116716437A
- Authority
- CN
- China
- Prior art keywords
- primer
- seq
- amplification
- tick
- pair
- 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
- 241000700605 Viruses Species 0.000 title claims abstract description 45
- 230000003321 amplification Effects 0.000 title claims abstract description 34
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 34
- 238000013412 genome amplification Methods 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 title claims abstract description 9
- 235000009347 chasteberry Nutrition 0.000 title abstract description 9
- 240000000059 Vitex cofassus Species 0.000 title abstract 2
- 241001643642 Viticis Species 0.000 claims abstract description 13
- 238000003757 reverse transcription PCR Methods 0.000 claims abstract description 7
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 6
- 238000012163 sequencing technique Methods 0.000 claims abstract description 4
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 241000532412 Vitex Species 0.000 claims description 9
- 108020004707 nucleic acids Proteins 0.000 claims description 4
- 102000039446 nucleic acids Human genes 0.000 claims description 4
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 235000001667 Vitex agnus castus Nutrition 0.000 claims 2
- 210000000051 wattle Anatomy 0.000 claims 1
- 238000013461 design Methods 0.000 abstract description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 108700026244 Open Reading Frames Proteins 0.000 abstract 1
- 241000238876 Acari Species 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 241000282412 Homo Species 0.000 description 2
- 206010037660 Pyrexia Diseases 0.000 description 2
- 108020000999 Viral RNA Proteins 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 244000052637 human pathogen Species 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 description 1
- 101710197658 Capsid protein VP1 Proteins 0.000 description 1
- 201000003075 Crimean-Congo hemorrhagic fever Diseases 0.000 description 1
- 241000712471 Dhori virus Species 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 101710144127 Non-structural protein 1 Proteins 0.000 description 1
- 101710144128 Non-structural protein 2 Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 102100022648 Reticulon-2 Human genes 0.000 description 1
- 102100031776 SH2 domain-containing protein 3A Human genes 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 101710106388 Structural protein VP1 Proteins 0.000 description 1
- 208000004374 Tick Bites Diseases 0.000 description 1
- 208000004006 Tick-borne encephalitis Diseases 0.000 description 1
- 241000710771 Tick-borne encephalitis virus Species 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 244000248021 Vitex negundo Species 0.000 description 1
- 235000010363 Vitex negundo Nutrition 0.000 description 1
- 208000035472 Zoonoses Diseases 0.000 description 1
- 244000037640 animal pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 244000309457 enveloped RNA virus Species 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 206010048282 zoonosis Diseases 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a tick-derived vitex virus genome amplification primer group, an amplification method and application, and belongs to the technical field of biology. The invention uses 12 pairs of primers as the upstream and downstream primers of the amplification primers, uses the Viticis negundo RNA template extracted from the tick sample to carry out RT-PCR amplification to obtain amplification products respectively, carries out nucleic acid sequence sequencing on the obtained amplification products, and then splices and compares the amplification products to obtain the tick-derived Viticis negundo virus genome sequence. The invention designs a sectional amplification strategy according to the open reading frame included in the genome aiming at the characteristics of the tick-derived Jinggar virus genome and referring to other known Jinggar virus genome sequences, and designs corresponding amplification primers according to a conserved region, thereby effectively amplifying the tick-derived Jinggar virus genome sequences; the invention lays a material foundation for tick-derived Viticis negundo virus detection and genome amplification requirements.
Description
Technical Field
The invention relates to a tick source vitex negundo virus genome amplification primer group, an amplification method and application, and belongs to the technical field of biology.
Background
Ticks are a hematophagous parasite belonging to the phylum acarina and the general family of ticks. Ticks are important human and animal pathogen vectors that can transmit pathogens by biting the host, severely threatening human health and production in animal husbandry; various viruses can be transmitted through tick bites, and mainly include fever with thrombocytopenia syndrome virus, crimedes Congo hemorrhagic fever virus, forest encephalitis virus and other zoonotic diseases. Such as tick borne encephalitis virus, can cause serious neurological diseases, i.e., tick borne encephalitis, which can cause damage to the central nervous system of animals and humans. These tick-borne viruses pose a great threat to public health safety.
The Jinmen tick virus is a single-strand positive-strand enveloped RNA virus, and the genome consists of four segments, wherein segment 1 and segment 3 respectively encode nonstructural proteins NSP1 and NSP2, segment 2 encodes structural protein VP1, and segment 4 respectively encodes structural proteins VP2 and VP3; the virus can be transmitted by various ticks, and the vitex tick virus is detected in a human body with fever symptoms after the ticks bite, which indicates that the virus can be the cause of the symptoms; the vitex tick virus may be a new tick-borne human pathogen, potentially pathogenic to humans.
Ticks are vectors of many viruses, often causing significant harm to human and animal health; the vegetation types of China are rich and various, the agriculture and animal industry is the main part in many places, and tick species groups are rich; therefore, designing primers for the tick carried Jingguan tick virus, establishing a genome amplification method, and carrying out gene analysis on an amplification sequence is an important basis for understanding the Jingguan tick virus, is favorable for grasping the epidemic trend and variation condition of the virus, and has important significance for related research of the Jingguan tick virus.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide a tick-derived vitex tick virus genome amplification method with high specificity and sensitivity.
The tick-derived Jinmen tick virus genome amplification primer set comprises 12 pairs of amplification primers, and the names and sequences of the 12 pairs of universal primers are as follows:
primer pair 1:
the forward primer 1JMTV-1-F is shown as SEQ ID NO. 1: 5'-TTACTGCAAGTGCAAAGGTTAAGG-3';
the reverse primer 1JMTV-1-R is shown as SEQ ID NO. 2: 5'-GTGTTGTTGAGGTGAGTTGCCAT-3';
primer pair 2:
the forward primer 2JMTV-2-F is shown as SEQ ID NO: 3: 5'-TCTATAGCAGAGATGCATATGGAA-3';
the reverse primer 2JMTV-2-R is shown as SEQ ID NO. 4: 5'-CATTTCTTCGTCCTCCGCTA-3';
3 rd pair of primers:
the forward primer 3JMTV-3-F is shown as SEQ ID NO. 5: 5'-ACCAGGCAGGTGAACAAAGGAAG-3';
the reverse primer 3JMTV-3-R is shown as SEQ ID NO. 6: 5'-TTGTGTTCATGTACGGTGGCGCTAA-3';
primer pair 4:
the forward primer 4JMTV-4-F is shown as SEQ ID NO. 7: 5'-TACCGCTGACTCCCCAAGGAAG-3';
the reverse primer 4JMTV-4-R is shown as SEQ ID NO. 8: 5'-CACCGGTCTGGTGTAGATGGAGTAC-3';
primer pair 5:
the forward primer 5JMTV-5-F is shown as SEQ ID NO: 9: 5'-ATATGCTCTACACATGAACACGAC-3';
the reverse primer 5JMTV-5-R is shown as SEQ ID NO. 10: 5'-GCGACCACTGTTGACCATGTG-3';
primer pair 6:
the forward primer 6JMTV-6-F is shown as SEQ ID NO. 11: 5'-CAACAACTTACCAACAGTGCTTGTG-3';
the reverse primer 6JMTV-6-R is shown as SEQ ID NO. 12: 5'-ACGCATGCGGCCATAACC-3';
primer pair 7:
the forward primer 7JMTV-7-F is shown as SEQ ID NO. 13: 5'-CCTCGCGCTAGACTCACTTTACAGA-3';
the reverse primer 7JMTV-7-R is shown as SEQ ID NO. 14: 5'-GCTAGTGCGAAGGTGCGATCTC-3';
primer pair 8:
the forward primer 8JMTV-8-F is shown as SEQ ID NO. 15: 5'-GATCGAGAACAGCCAGAGTGCAG-3';
the reverse primer 8JMTV-8-R is shown as SEQ ID NO. 16: 5'-GACAAGGTAGTGGTACTGTTGGC-3';
primer pair 9:
forward primer 9JMTV-9-F: as shown in SEQ ID NO. 17: 5'-TTCAGGGACTACCCTAGAGGGCTG-3';
the reverse primer 9JMTV-9-R is shown as SEQ ID NO. 18: 5'-GCTAGCCGCAACCTAGTCATTGC-3';
primer of the 10 th pair:
the forward primer 10JMTV-10-F is shown as SEQ ID NO. 19: 5'-GAGTTGCAAGTGCMATAGCTCG-3';
the reverse primer 10JMTV-10-R is shown as SEQ ID NO. 20: 5'-TTGAARGCCAGSGACACCAC-3';
11 th pair of primers:
the forward primer 11JMTV-11-F is shown as SEQ ID NO. 21: 5'-GCCTGATACGACAGTATCTCGAGAGAAG-3';
the reverse primer 11JMTV-11-R is shown as SEQ ID NO. 22: 5'-TTTCCTGCTATGACGACAAGCCA-3';
primer pair 12:
the forward primer 12JMTV-12-F is shown as SEQ ID NO. 23: 5'-TCTTCAGCCTTGCGGTCTACAC-3';
the reverse primer 12JMTV-12-R is shown as SEQ ID NO. 24: 5'-AGCACTTGCCATCTCCATGCTC-3'.
By using the 12 pairs of tick-derived Jingguan tick virus genome amplification primer groups, the tick-derived Jingguan tick virus genome sequence information can be obtained through PCR amplification, sequencing and splicing, and the specific technical scheme comprises the following steps:
and respectively taking the primer pairs 1-12 as upstream and downstream primers for amplification, carrying out RT-PCR amplification on the Viticis negundo RNA template extracted from the tick sample to respectively obtain amplification products, then carrying out nucleic acid sequence sequencing on the amplification products of each pair of primers, and then splicing and comparing to obtain the tick-derived Viticis negundo virus genome sequence.
Further, the RT-PCR reaction system is as follows:
the reaction conditions are as follows: the reaction systems of the 12 pairs of primers are all reversed for 30min at 50 ℃; pre-denaturation at 95 ℃ for 5min; then, the temperature is 95 ℃ for 30s; 30s at 50 ℃;72 ℃ for 1min30s; 39 cycles total and finally extended for 7min at 72 ℃.
The invention has the beneficial effects that:
(1) The invention designs corresponding amplification primers aiming at the characteristics of tick-derived Viticis negundo virus genome and referring to the known other tick-derived Viticis negundo virus genome sequences, and finally screens out 12 primer pairs through multiple attempts to effectively amplify the tick-derived Viticis negundo virus genome sequences; the invention lays a material foundation for tick-derived Viticis negundo virus detection and genome amplification requirements.
(2) The one-step RT-PCR designed by the invention is used for amplifying tick-derived Jinmen tick virus genome, and has the characteristics of simple and rapid operation, low cost, high sensitivity and the like; the genome of the tick-derived Jinmen tick virus can be obtained by one-step experiment, and the occurrence of pollution is reduced as much as possible.
Drawings
FIG. 1 is an electrophoretogram of tick-derived Viticis negundo virus genome amplification products.
Running the PCR amplified product by using 2% agarose gel; wherein M:2000bpmarker;1-12: the electrophoresis results of the 12 product fragments amplified by the 12 pairs of primer pairs of the tick-derived Jinmen tick virus genome are from left to right.
Detailed Description
The invention will be described in further detail with reference to the drawings and the specific embodiments, but the scope of the invention is not limited to the description.
The experimental methods in the embodiments of the present invention are conventional methods unless otherwise specified.
The percentages in the examples below are mass percentages unless otherwise indicated.
Example 1
(1) The virus sample treatment adopts tick source vitex virus positive sample, tick sample is taken, PBS buffer solution is added into 1/10 (w/v), and the mixture is ground for 5 minutes by a grinder; taking out the grinding fluid, centrifuging for 30min at 4 ℃ and 10,000Xg, and sucking all the supernatant for later use.
(1) Viral RNA extraction: the method adopts a viral RNA/DNA nucleic acid extraction box (MiniBESTViralRNA/DNAExtractionkit, cat. # 9766) which is proposed by Takara Bio-engineering (Dalian) Inc., and comprises the following specific experimental steps according to the instruction of the kit, and preferably adjusted according to the actual experimental conditions:
(1) 200. Mu.l of the obtained supernatant was taken, 200. Mu.l of BufferVGB, 20. Mu.l of Proteina seK and 1.0. Mu.l of Carrier rRNA were added thereto, thoroughly mixed, incubated in a 56℃water bath for 10 minutes, 200. Mu.l of 96-100% ethanol was added to the lysate, and thoroughly sucked and mixed.
(2) SpinColumn was mounted on a CollectionTube, the previous solution was transferred to SpinC column, centrifuged at 12000rpm for 2 minutes, and the filtrate was discarded.
(3) Mu.l of BufferRWA was added to the spinColumbn and centrifuged at 12000rpm for 1min, and the filtrate was discarded.
(4) 700 μl of BufferRWB was added to spinColumbn, centrifuged at 12000rpm for 1min, and the filtrate was discarded; confirming in advance whether 100% ethanol with a specified volume is added in BufferRWB; and the bufferrfb is added from the periphery of the SpinColumn pipe wall, so that the salt adhered to the pipe wall can be completely washed.
(5) Repeating the operation step (4).
(6) SpinColumn was mounted on a CollectionTube and centrifuged at 12,000rpm for 2 minutes.
(7) Place spinColumbn on New 1.5ml RNasetreeclectic tube with 30-50. Mu.l RNasebreedH added in the center of SpinColumbn film 2 O, standing for 5 minutes at room temperature; RNasebreedH was used when RNA on the preparation membrane was eluted 2 O。
(8) RNA was eluted using centrifugation at 12000rpm for 2 minutes.
It is noted that repeating step (8) for the final higher nucleic acid concentration increases the elution efficiency and increases the nucleic acid concentration.
(3) Design and Synthesis of amplification primers for tick-derived Viticis negundo viral genome: downloading tick-derived Jinmen tick virus strains from a GenBank database as reference sequences, then carrying out sequence comparison by using ClustalW (version 2.0), selecting a conserved region sequence, analyzing GC content, tm value, hairpin structure and primer dimer by PrimerSelect software, and indicating that the primer is initially designed to be complete if all the sequences are qualified; then, the optimal primer is determined by screening and then sent to Beijing qingke biotechnology Co., ltd for primer synthesis; finally, 12 pairs of amplification primers are obtained, specifically:
primer pair 1:
the forward primer 1JMTV-1-F is shown as SEQ ID NO. 1: 5'-TTACTGCAAGTGCAAAGGTTAAGG-3';
the reverse primer 1JMTV-1-R is shown as SEQ ID NO. 2: 5'-GTGTTGTTGAGGTGAGTTGCCAT-3';
primer pair 2:
the forward primer 2JMTV-2-F is shown as SEQ ID NO: 3: 5'-TCTATAGCAGAGATGCATATGGAA-3';
the reverse primer 2JMTV-2-R is shown as SEQ ID NO. 4: 5'-CATTTCTTCGTCCTCCGCTA-3';
3 rd pair of primers:
the forward primer 3JMTV-3-F is shown as SEQ ID NO. 5: 5'-ACCAGGCAGGTGAACAAAGGAAG-3';
the reverse primer 3JMTV-3-R is shown as SEQ ID NO. 6: 5'-TTGTGTTCATGTACGGTGGCGCTAA-3';
primer pair 4:
the forward primer 4JMTV-4-F is shown as SEQ ID NO. 7: 5'-TACCGCTGACTCCCCAAGGAAG-3';
the reverse primer 4JMTV-4-R is shown as SEQ ID NO. 8: 5'-CACCGGTCTGGTGTAGATGGAGTAC-3';
primer pair 5:
the forward primer 5JMTV-5-F is shown as SEQ ID NO: 9: 5'-ATATGCTCTACACATGAACACGAC-3';
the reverse primer 5JMTV-5-R is shown as SEQ ID NO. 10: 5'-GCGACCACTGTTGACCATGTG-3';
primer pair 6:
the forward primer 6JMTV-6-F is shown as SEQ ID NO. 11: 5'-CAACAACTTACCAACAGTGCTTGTG-3';
the reverse primer 6JMTV-6-R is shown as SEQ ID NO. 12: 5'-ACGCATGCGGCCATAACC-3';
primer pair 7:
the forward primer 7JMTV-7-F is shown as SEQ ID NO. 13: 5'-CCTCGCGCTAGACTCACTTTACAGA-3';
the reverse primer 7JMTV-7-R is shown as SEQ ID NO. 14: 5'-GCTAGTGCGAAGGTGCGATCTC-3';
primer pair 8:
the forward primer 8JMTV-8-F is shown as SEQ ID NO. 15: 5'-GATCGAGAACAGCCAGAGTGCAG-3';
the reverse primer 8JMTV-8-R is shown as SEQ ID NO. 16: 5'-GACAAGGTAGTGGTACTGTTGGC-3';
primer pair 9:
forward primer 9JMTV-9-F: as shown in SEQ ID NO. 17: 5'-TTCAGGGACTACCCTAGAGGGCTG-3';
the reverse primer 9JMTV-9-R is shown as SEQ ID NO. 18: 5'-GCTAGCCGCAACCTAGTCATTGC-3';
primer of the 10 th pair:
the forward primer 10JMTV-10-F is shown as SEQ ID NO. 19: 5'-GAGTTGCAAGTGCMATAGCTCG-3';
the reverse primer 10JMTV-10-R is shown as SEQ ID NO. 20: 5'-TTGAARGCCAGSGACACCAC-3';
11 th pair of primers:
the forward primer 11JMTV-11-F is shown as SEQ ID NO. 21: 5'-GCCTGATACGACAGTATCTCGAGAGAAG-3';
the reverse primer 11JMTV-11-R is shown as SEQ ID NO. 22: 5'-TTTCCTGCTATGACGACAAGCCA-3';
primer pair 12:
the forward primer 12JMTV-12-F is shown as SEQ ID NO. 23: 5'-TCTTCAGCCTTGCGGTCTACAC-3';
the reverse primer 12JMTV-12-R is shown as SEQ ID NO. 24: 5'-AGCACTTGCCATCTCCATGCTC-3'.
(4) Genome segment amplification (i.e., 12-segment amplification, the primers used are the 12 pairs of primers described above):
viral nucleic acid RT-PCR amplification PCR reaction system: 2X1stepbuffer 6. Mu.L, upstream primer (10. Mu.M) 1. Mu.L, downstream primer (10. Mu.M) 1. Mu.L, primeScript1stepenzymeMix 0.5. Mu. L, ddH 2 O3 mu L, RNATemplate mu L and the total reaction system is 12.5 mu L; the reaction conditions are as follows: the 12 pairs of primer reaction systems are as follows: reversing at 50 degrees for 30min; pre-denaturation at 95 ℃ for 5min; then, the temperature is 95 ℃ for 30s; 30s at 50 ℃;72 ℃ for 1min30s; 39 cycles total and finally extended for 7min at 72 ℃.
(4) Electrophoresis and PCR product sequence determination: taking 3 mu L of amplified products, adopting 2% agarose gel for electrophoresis, and observing the results through a gel imaging system; the amplification of the tick-derived JINGGAN virus genome was performed in the order of JMTV-1-F/JMTV-1-R, JMTV-2-F/JMTV-2-R, JMTV-3-F/JMTV-3-R, JMTV-4-F/JMTV-4-R, JMTV-5-F/JMTV-5-R, JMTV-6-F/JMTV-6-R, JMTV-7-F/JMTV-7-R, JMTV-8-F/JMTV-8-R, JMTV-9-F/JMTV-9-R, JMTV-10-F/JMTV-10-R, JMTV-11-F/JMTV-11-R, JMTV-12-F/JMTV-12-R in the order of 1268bp, 830bp, 1300bp, 760bp, 1309bp, 1028bp, 961bp, 984bp, 842bp, 1289bp, 832bp, and the size range, as indicated by the result of the electrophoresis using the primers shown in FIG. 1.
(5) Splicing and aligning genome sequences: splicing the sequence fragments amplified by the 12 pairs of amplification primers on the tick-derived vitex virus to obtain a genome sequence; the Gene sequences of the 4 segments are submitted to Gene Bank for BLAST analysis, and the result shows that the coverage of the obtained genome sequence and the tick source vitex virus genome sequence reaches 100%, and the similarity reaches more than 94%.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (4)
1. A tick-derived, chaste tree tick virus genome amplification primer set, characterized in that: the amplification primer set comprises the following 12 pairs of amplification primers:
primer pair 1:
the forward primer 1JMTV-1-F is shown as SEQ ID NO. 1: 5'-TTACTGCAAGTGCAA AGGTTAAGG-3';
the reverse primer 1JMTV-1-R is shown as SEQ ID NO. 2: 5'-GTGTTGTTGAGGTGA GTTGCCAT-3';
primer pair 2:
the forward primer 2JMTV-2-F is shown as SEQ ID NO: 3: 5'-TCTATAGCAGAGATGC ATATGGAA-3';
the reverse primer 2JMTV-2-R is shown as SEQ ID NO. 4: 5'-CATTTCTTCGTCCTCC GCTA-3';
3 rd pair of primers:
the forward primer 3JMTV-3-F is shown as SEQ ID NO. 5: 5'-ACCAGGCAGGTGAAC AAAGGAAG-3';
the reverse primer 3JMTV-3-R is shown as SEQ ID NO. 6: 5'-TTGTGTTCATGTACGG TGGCGCTAA-3';
primer pair 4:
the forward primer 4JMTV-4-F is shown as SEQ ID NO. 7: 5'-TACCGCTGACTCCCC AAGGAAG-3';
the reverse primer 4JMTV-4-R is shown as SEQ ID NO. 8: 5'-CACCGGTCTGGTGTA GATGGAGTAC-3';
primer pair 5:
the forward primer 5JMTV-5-F is shown as SEQ ID NO: 9: 5'-ATATGCTCTACACATG AACACGAC-3';
the reverse primer 5JMTV-5-R is shown as SEQ ID NO. 10: 5'-GCGACCACTGTTGAC CATGTG-3';
primer pair 6:
the forward primer 6JMTV-6-F is shown as SEQ ID NO. 11: 5'-CAACAACTTACCAA CAGTGCTTGTG-3';
the reverse primer 6JMTV-6-R is shown as SEQ ID NO. 12: 5'-ACGCATGCGGCCAT AACC-3';
primer pair 7:
the forward primer 7JMTV-7-F is shown as SEQ ID NO. 13: 5'-CCTCGCGCTAGACTC ACTTTACAGA-3';
the reverse primer 7JMTV-7-R is shown as SEQ ID NO. 14: 5'-GCTAGTGCGAAGGT GCGATCTC-3';
primer pair 8:
the forward primer 8JMTV-8-F is shown as SEQ ID NO. 15: 5'-GATCGAGAACAGCC AGAGTGCAG-3';
the reverse primer 8JMTV-8-R is shown as SEQ ID NO. 16: 5'-GACAAGGTAGTGGT ACTGTTGGC-3';
primer pair 9:
forward primer 9JMTV-9-F: as shown in SEQ ID NO. 17: 5'-TTCAGGGACTACCC TAGAGGGCTG-3';
the reverse primer 9JMTV-9-R is shown as SEQ ID NO. 18: 5'-GCTAGCCGCAACCTA GTCATTGC-3';
primer of the 10 th pair:
the forward primer 10JMTV-10-F is shown as SEQ ID NO. 19: 5'-GAGTTGCAAGTGC MATAGCTCG-3';
the reverse primer 10JMTV-10-R is shown as SEQ ID NO. 20: 5'-TTGAARGCCAGSG ACACCAC-3';
11 th pair of primers:
the forward primer 11JMTV-11-F is shown as SEQ ID NO. 21: 5'-GCCTGATACGACA GTATCTCGAGAGAAG-3';
the reverse primer 11JMTV-11-R is shown as SEQ ID NO. 22: 5'-TTTCCTGCTATGAC GACAAGCCA-3';
primer pair 12:
the forward primer 12JMTV-12-F is shown as SEQ ID NO. 23: 5'-TCTTCAGCCTTGCG GTCTACAC-3';
the reverse primer 12JMTV-12-R is shown as SEQ ID NO. 24: 5'-AGCACTTGCCATC TCCATGCTC-3'.
2. A method of amplification of tick-derived wattle virus genome based on the amplification primer set of claim 1, characterized in that: and respectively taking the primer pairs 1-12 as upstream and downstream primers for amplification, carrying out RT-PCR amplification on the Viticis negundo RNA template extracted from the tick sample to respectively obtain amplification products, and then carrying out nucleic acid sequencing on the amplification products of each pair of primers, and then splicing and comparing to obtain the Viticis negundo genome sequence.
3. The method according to claim 2, characterized in that: the RT-PCR reaction system is as follows:
the reaction conditions are as follows: the reaction systems of the 12 pairs of primers are all reversed for 30min at 50 ℃; pre-denaturation at 95 ℃ for 5min; then, the temperature is 95 ℃ for 30s; 30s at 50 ℃; for 39 cycles at 72℃in30s, and for 7min at 72 ℃.
4. Use of a primer pair according to claim 1 for amplification to give a tick-derived, chaste tree virus genomic sequence.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310651946.3A CN116716437A (en) | 2023-06-05 | 2023-06-05 | Tick-derived vitex virus genome amplification primer group, amplification method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310651946.3A CN116716437A (en) | 2023-06-05 | 2023-06-05 | Tick-derived vitex virus genome amplification primer group, amplification method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116716437A true CN116716437A (en) | 2023-09-08 |
Family
ID=87874538
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310651946.3A Pending CN116716437A (en) | 2023-06-05 | 2023-06-05 | Tick-derived vitex virus genome amplification primer group, amplification method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116716437A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016022958A2 (en) * | 2014-08-07 | 2016-02-11 | The Trustees Of Columbia University In The City Of New York | Tick-associated virus sequences and uses thereof |
| CN116042872A (en) * | 2022-11-25 | 2023-05-02 | 海南省人民医院 | Composition for combined detection of eight pathogens such as TBEV and detection method |
-
2023
- 2023-06-05 CN CN202310651946.3A patent/CN116716437A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016022958A2 (en) * | 2014-08-07 | 2016-02-11 | The Trustees Of Columbia University In The City Of New York | Tick-associated virus sequences and uses thereof |
| CN116042872A (en) * | 2022-11-25 | 2023-05-02 | 海南省人民医院 | Composition for combined detection of eight pathogens such as TBEV and detection method |
Non-Patent Citations (2)
| Title |
|---|
| YULI ZHANG等: "Identification of Jingmen tick virus (JMTV) in Amblyomma testudinarium from Fujian Province, southeastern China", PARASITES & VECTORS, vol. 15, pages 4 - 5 * |
| ZHU-MEI YU等: "Identification and characterization of Jingmen tick virus in rodents from Xinjiang, China", INFECTION, GENETICS AND EVOLUTION, vol. 84, pages 2 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Berhanu et al. | Molecular characterization of partial fusion gene and C-terminus extension length of haemagglutinin-neuraminidase gene of recently isolated Newcastle disease virus isolates in Malaysia | |
| Sabanadzovic et al. | Properties and detection of two cryptoviruses from pepper (Capsicum annuum) | |
| Ohlemeyer et al. | Major capsid protein gene sequence analysis of the Santee-Cooper ranaviruses DFV, GV6, and LMBV | |
| Goszczynski | Divergent molecular variants of Grapevine virus B (GVB) from corky bark (CB)-affected and CB-negative LN33 hybrid grapevines | |
| Rodrigues et al. | Phylogeny and recombination analysis of Brazilian yellow passion fruit isolates of Cowpea aphid-borne mosaic virus: origin and relationship with hosts | |
| CN113736750B (en) | Gata virus strain and application thereof | |
| Aydin et al. | A pilot study on feline astrovirus and feline panleukopenia virus in shelter cats in Erzurum, Turkey | |
| Mishra et al. | Sequence diversity studies of papaya ringspot virus isolates in South India reveal higher variability and recombination in the 5′-terminal gene sequences | |
| CN116716437A (en) | Tick-derived vitex virus genome amplification primer group, amplification method and application | |
| Nida'M et al. | Further complexity of the genus Crinivirus revealed by the complete genome sequence of Lettuce chlorosis virus (LCV) and the similar temporal accumulation of LCV genomic RNAs 1 and 2 | |
| CN111575307A (en) | Armored RNA standard substance containing Hancheng virus G2 protein gene and preparation method thereof | |
| Moradi et al. | First report of Narcissus latent virus infecting iris in Iran | |
| CN108796128B (en) | GII.8 type norovirus genome amplification primer and amplification method | |
| CN109485714B (en) | TLK protein and application thereof in shrimp and crab antiviral strain breeding | |
| Feng et al. | Serotype and VP1 gene sequence of a foot-and-mouth disease virus from Hong Kong (2002) | |
| CN108796129B (en) | GI.Pb/GI.6 recombinant norovirus genome amplification primer and amplification method | |
| Hu et al. | Identification of a novel Getah virus by Virus-Discovery-cDNA random amplified polymorphic DNA (RAPD) | |
| CN113755565A (en) | Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying African swine fever wild viruses and vaccine strains | |
| Haokip et al. | Characterization of Capsicum chlorosis virus infecting chilli (Capsicum annuum. L) in southern India | |
| Hsieh et al. | Detection of Ranavirus by PCR and in situ hybridization in the American bullfrog (Rana catesbeiana) in Taiwan | |
| CN108796130B (en) | GII.2 type norovirus genome amplification primer and amplification method | |
| Hu et al. | Complete genome and phylogenetic analysis of bovine papillomavirus type 15 in Southern Xinjiang dairy cow | |
| CN108823331B (en) | GII.6 type norovirus genome amplification primer and amplification method | |
| CN111321246A (en) | Canine distemper virus nano PCR detection kit and application thereof | |
| Moradi et al. | Identification and molecular analysis of sugarcane mosaic virus (SCMV) in Mazandaran province |
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 |