LU501782B1 - Pcr primer, reagent or kit for identifying or assisting in identifying freshwater fish species and identification method - Google Patents
Pcr primer, reagent or kit for identifying or assisting in identifying freshwater fish species and identification method Download PDFInfo
- Publication number
- LU501782B1 LU501782B1 LU501782A LU501782A LU501782B1 LU 501782 B1 LU501782 B1 LU 501782B1 LU 501782 A LU501782 A LU 501782A LU 501782 A LU501782 A LU 501782A LU 501782 B1 LU501782 B1 LU 501782B1
- Authority
- LU
- Luxembourg
- Prior art keywords
- identifying
- pcr primer
- fish
- species
- cyprinidae
- Prior art date
Links
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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- 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]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present disclosure provides a new PCR primer (16s 200) for identifying or assisting in identifying freshwater fish species, and the nucleotide sequence of the primer is SEQ ID NO.1 in a sequence table. The PCR primer is used for metabarcoding of eDNA of a fish mitochondrial DNA sequence. The PCR primer is used for PCR amplification of eDNA in a water body. Based on a high-throughput sequencing method, the problem of difficulty in obtaining effective species-specific primers when there are many related species in the water body can be solved effectively. Based on the environmental DNA metabarcoding technology, effective technical support is provided for fish taxonomy research and diversity protection thereof. A method for identifying the freshwater fish species by the PCR primer has the advantages of being short in time, simple in operation and strong in specificity.
Description
PCR PRIMER, REAGENT OR KIT FOR IDENTIFYING OR ASSISTING IN IDENTIFYING FRESHWATER FISH SPECIES AND IDENTIFICATION METHOD LUs01782
TECHNICAL FIELD The present disclosure relates to a PCR primer, a reagent or a kit for identifying or assisting in identifying freshwater fish species and an identification method and belongs to the field of molecular ecology.
BACKGROUND OF THE PRESENT INVENTION Due to environmental changes and human activities, global biodiversity is rapidly decreasing. Freshwater ecosystem is one of the most fragile ecosystems on the earth. Freshwater fish is an important part of the freshwater ecosystem. Due to climate changes, habitat degradation, biological invasion, overfishing and other factors, the diversity of freshwater fishes is rapidly decreasing. Accurate evaluation of responses of fish populations and aquatic communities to environmental changes and quantification of influences of changes in the structure and function of fish communities on aquatic ecosystems are very important for protecting freshwater ecosystems and fish diversity. The traditional fish monitoring technology mainly uses nets, fishing tackles, electric fishing gears, diving and other means to collect solid specimens on the spot. Disadvantages of such type of destructive sampling are obvious: for rare species, low-density species, especially early invasive species and fishes distributed in complex and dangerous habitats, sampling is difficult or even inoperable, and these methods are often time-consuming and laborious, which may harm target species or destroy the ecosystem at investigation sites.
Another common technique of fish monitoring is acoustic detection, which uses an echo sounder to evaluate statuses of fish resources in various water bodies. Through the study of echo imaging, and full combination with fish sampling of stratified trawls, the integral distribution is carried out, so as to obtain a conversion relation between the target intensity and fish body length, and finally estimate the quantity and distribution of the fish resources in waters. The results can better help to understand the quantity and distribution of the fishes compared with the fishing method. However, the technology cannot classify different fishes and accurately quantify the fishes, and needs to be based on a correct fishing model. At present, all fishing finders are modeled by foreign marine fishes as the standards, and there is a big doubt whether they are suitable for freshwater fishes in China. This technology is also limited by physical and chemical indexes of waters and weather conditions. In recent years, environmental DNA (eDNA) metabarcoding technology has attracted the attention of aquatic biology researchers because of its advantages of accurate identification of molecular biological means, no interference to target organisms and convenient sample collection.
Environmental DNA refers to the DNA released from an organism into the natural environment (such as air, water, soil, etc.), including the DNA in cells and DNA molecules LU501782 released outside cells after cell breakage. In recent years, the environmental DNA metabarcoding technology has attracted the attention of aquatic biology researchers because of the advantages of high efficiency, no interference to target organisms and convenient sample collection. The advantages of the DNA metabarcoding technology mainly include: (1) survey sensitivity is high; (2) sampling is convenient; (3) the technology is environment-friendly and free of no damage to investigated objects; (4) time and labor are saved, and cost of investigation is low; and (5) sampling time is less affected by natural factors such as weather. The application of environmental DNA metabarcoding technology in large aquatic organisms originated from the detection research of American bullfrogs by Ficetola in 2008. Since then, the qualitative detection research of aquatic organisms by the environmental DNA metabarcoding technology has been carried out, and good results have been achieved in monitoring of alien fish invasion, detection of rare fish species and investigation of fish community resources. For example, a water sample eDNA analysis method is used to monitor diffusion trends of Hypophthalmichthys molitrix and Hypophthalmichthys nobilis in Lake Michigan and its surrounding rivers, a water sample eDNA analysis method is used to analyze and track diffusion trends of the invasive species bluegill sunfish (Lepomis macrochirus) in Japan and its surrounding islands, etc. All these methods have achieved better monitoring results than traditional observation methods. Miya et al. designed universal primers for marine fishes by analyzing mitochondrial sequences of several marine fish species, and combined the application with a high-throughput sequencing technology, to verify its feasibility in environmental DNA metabarcoding method. However, in the subsequent research, it was found that the universal primers designed by foreign researchers for freshwater fishes were not ideal when used in domestic environment. The reason was that the composition of freshwater fish populations in China were primarily composed of closely-related carp (Cyprinidae) species, and the genetic relationship between fish species was relatively close. However, when the universal primers designed by foreign researchers were used for amplification comparison, the amplified fragment sequences of some fishes were the same, which could not realize accurate identification. Therefore, at present, it is impossible to give consideration to both accuracy and breadth during use of universal primers for freshwater fishes in China.
SUMMARY OF PRESENT INVENTION In order to overcome the above problems, based on DNA sequences of Chinese freshwater fishes, the present disclosure provides a new PCR primer (16s 200) for identifying or assisting in identifying freshwater fish species. The PCR primer is used for metabarcoding of eDNA of a fish mitochondrial DNA sequence. The PCR primer is used to for PCR amplification of eDNA in a water body. Based on a high-throughput sequencing method, the problem that it is difficult to obtain effective species-specific primers when there are many related species in the water can be solved effectively. Based on the environmental DNA metabarcoding technology, LU501782 effective technical support is provided for fish taxonomy research and diversity protection thereof. À method for identifying the freshwater fish species by the PCR primer has the advantages of being short in time, simple in operation and strong in specificity. A PCR primer for identifying or assisting in identifying freshwater fish species: the nucleotide sequence of the primer is SEQ ID NO.1 in a sequence table.
Further, the PCR primer is used for metabolic coding of eDNA of a fish mitochondrial DNA sequence.
A reagent or kit for identifying or assisting in identifying freshwater fish species comprises the PCR primer.
A method for identifying or assisting in identifying freshwater fish species by the PCR primer or the reagent or kit described above comprises: using the PCR primer for PCR amplification of a water sample to be detected, sequencing an amplified product, correcting or editing a sequence obtained by sequencing, submitting the sequence to a database for comparison, and identifying the fish species of the water sample according to a DNA sequence similarity.
Further, in terms of 25 ul, an amplification system comprises the following components: 2.5 ul of 10 x ExTaq Buffer (Mg?*Plus) (20 mM), 2 ul of dNTP Mixture (2.5 mM for each), 1 ul of each of forward and reverse primers (10 umol/l), 0.2 pl of TaKaRa TagHS, 2 ul of DNA template, and 16.3 ul of ddH2O.
Further, an amplification procedure is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 60.3°C for 40 s, extension at 72°C for 1 min, for 35 cycles; and extension for 10 min after 71°C.
Further, in the amplification procedure, an optimum annealing temperature of the PCR primer is 60.3°C.
Compared with nuclear DNA, mitochondrial DNA of fish has the characteristics of being small in molecule, simple in structure, easy in extraction and maternal in inheritance. Therefore, mitochondrial DNA fragments are selected as molecular markers for analyzing fish population structures and species classification and identification. Mitochondrial DNA sequences of 62 species of fish are downloaded from NCBI (see Table 1 for serial numbers and corresponding Latin names). After comparison of the sequences, universal primers are designed based on a hypervariable region of the 16S rRNA gene. In addition, the designed primers are compared with human mitochondrial DNA on NCBI, and the primers that can amplify human mitochondrial DNA are eliminated, which are synthesized by Sangon Biotech (Shanghai) Co., Ltd. Sequences of the primers and probes obtained are as follows (Table 2):
Table 1 Accession No. of fish mitochondrial genes used in designing | U50178 environmental DNA universal primers
~ Family Species Accession No. _
~ Bagridae =~ Tachyswusfulvidraco ~~ MH192350 — Cobitidae Paramisgurnusdabryanus KJ027397 Cobitidae Misgurnusanguillicaudatus KM186181 Cobitidae Cobitissinensis AY526868 Cyprinidae Tincatinca AB218686 Cyprinidae Carassiuscuvieri AP011237 Cyprinidae Hypophthalmichthysmolitrix EU315941 Cyprinidae Hemiculterellasauvagei KP316066 Cyprinidae Abbottinarivularis MK852690 Cyprinidae Hemiculterbleekeri NC_029831 Cyprinidae Parabramispekinensis MK421554 Cyprinidae Rhodeuslighti NC_024885 Cyprinidae Hemiculterleucisculus KF956522 Cyprinidae Ctenopharyngodonidella EU391390 Cyprinidae Pseudorasboraelongata KF245485 Cyprinidae Saurogobiodumerili NC_022187 Cyprinidae Hemibarbuslongirostris DQ347952 Cyprinidae Squaliobarbuscurriculus KP731975 Cyprinidae Acheilognathusmacropterus KJ499466 Cyprinidae Chanodichthysdabryi NC_021418 Cyprinidae Sinibramamacrops NC_020013 Cyprinidae Squaliduswolterstorffi NC_022190 Cyprinidae Megalobramaskolkovii KJ630486 Cyprinidae Microphysogobiofukiensis KJ933414 Cyprinidae Elopichthysbambusa NC_024834 Cyprinidae Rhodeusocellatus KT004415 Cyprinidae Acrossocheilusfasciatus NC_023378 Cyprinidae Spinibarbushollandi NC_026129 Cyprinidae Ochetobiuselongatus KM400625 Cyprinidae Sarcocheilichthysnigripinnis KJ997940 Cyprinidae Cultererythropterus NC_024749 Cyprinidae Xenocyprisdavidi KF039718 Cyprinidae Sarcocheilichthyssinensis KF177390 Cyprinidae Huigobiochenhsienensis NC_032291
Cyprinidae Microphysogobiotafangensis KF857260 LU501782 Cyprinidae Sarcocheilichthyskiangsiensis KY779851 Cyprinidae Phoxinusoxycephalus NC_027273 Cyprinidae Carassiusauratus KJ874430 Cyprinidae Zaccoplatypus NC_023105 Cyprinidae Microphysogobiokiatingensis NC_037402 Cyprinidae Cyprinuscarpio KU159761 Cyprinidae Pseudorasboraparva JF802126 Cyprinidae Opsariichthysbidens DQ367044 Cyprinidae Cultermongolicus AP009060 Cyprinidae Pseudolaubucaengaulis KC429670 Cyprinidae Culteralburnus KX244762 Cyprinidae Mylopharyngodonpiceus EU979307 Cyprinidae Saurogobiodabryi KF612272 Cyprinidae Pseudobramasimoni NC_022852 Cyprinidae Belligobionummifer NC_023975 Cyprinidae Toxabramisswinhonis NC_029249 Cyprinidae Pseudogobiovaillanti NC_032292 Cyprinidae Megalobramaamblycephala AP011219 Cyprinidae Acrossocheiluswenchowensis NC_020145 Cyprinidae Sarcocheilichthysvariegatus NC_046944 Cyprinidae Plagiognathopsmicrolepis NC_022711 Cyprinidae Acanthorhodeuschankaensis NC_023101 Cyprinidae Xenocyprisargentea AP009059 Cyprinidae Carassiusgibelio KX505166 Cyprinidae Hypophthalmichthysnobilis EU343733 Cyprinidae Spinibarbussinensis NC_022465 Cyprinidae Aphyocyprischinensis NC_008650
BE LUSO1782 Table 2 Sequence table of general primers for fish ~~ Reaction conditions Lengthof Sequence amplified fragment ~~ predenaturatonat 94°C for 3 min; 16s 200 F: denaturation at 94°C 5'-TGAGTATGGGAGACAGAAAAGGTTC-3' for 30 s; annealing at
60.3°C for 40 s: 255bp 16s 200 R: extension at 72°C for 1 5-CTTTTGCCACAGAGACGGGT-3’ min; for 35 cycles; extension for 10 min after 71°C The specific method for identifying or assisting in identifying freshwater fish species is as follows: (1) Sample DNA collection According to the size of a water area, a sample area to be investigated is set, a columnar water layer at a collection sample point is collected for mixing, refrigerated and brought back to the laboratory. Within 24 hours, the water sample is filtered by suction. After a filtered filter membrane is put into a sterilization centrifuge tube, the membrane is immediately put into a refrigerator at -20°C for later use. The filter membrane aperture is 1.2 um. As for the obtained samples, the kit QlAamp DNA MicroKit (Qiagen GmbH, Hilden, Germany) produced by QIAGEN Company or other similar kits (such as 1. Kit DNeasyBlood and Tissue Kit (Qiagen GmbH, Hilden, Germany); 2. Kit Power water Stereoex DNA Isolation Kit (Mobio, CA, etc.) is used to extract all environmental DNA in the filter membrane. The extracted DNA is stored at - 20°C for later use. (2) DNA amplification and high-throughput sequencing The extracted environmental DNA samples are subjected to PCR amplification using the synthesized primer 16s 200. Sample reaction system of 25 ul: 2.5 pl of 10xExTaq Buffer (Mg**Plus) (20 mM), 2 ul of dNTP Mixture (2.5 mM for each), 1 ul of each of forward and reverse primers (10 umol/l), 0.2 ul of TaKaRa TagHS, 2 ul of DNA template, and 16.3 pl of ddH2O. Reaction conditions: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 60.3°C for 40 s, extension at 72°C for 1 min, for 35 cycles; and extension for 10 min after 71°C. PCR products are detected by 1% gel electrophoresis, and the concentration is determined by NanoDrop™2000 spectrophotometer (Thermo FisherScience, Waltham, MA, USA). After testing, qualified samples are selected for high-throughput sequencing. (3) Data analysis
After sequencing, firstly, quality control and optimization of data are carried out, then LU501782 effective sequences of all the samples are clustered by OTUs (OperationalTaxonomicUnits Units) with the identity of 97%. Then species annotation of the sequences of OTUs is carried out according to the database of Genebank and the self-built library of the laboratory.
The present disclosure has the beneficial effects that: (1) Compared with existing fishing methods and hydroacoustic methods, the present disclosure greatly improves feasibility of sample detection because of the advantages of accurate identification of molecular biological means, no interference to target organisms and convenient sample collection. Compared with the existing DNA barcode technology, the technology platform used is completely consistent. More importantly, the present disclosure solves the problem that an existing DNA barcode is difficult to identify some fishes with close genetic relationships in China. Therefore, the DNA barcode and an application method thereof established by the present disclosure are important supplements to the existing DNA barcode technology.
(2) The present disclosure is based on the environmental DNA monitoring technology, does not depend on capture of fish species, and only needs to collect water samples to analyze existence and quantity of species. The sampling method is simple, and fish resources can be protected to the maximum extent. At the same time, this method is more effective than traditional fishing survey for some species which are less distributed and difficult to catch.
(3) Based on the DNA sequences of freshwater fishes in China, the present disclosure provides a new PCR primer (16s 200) used for metabolic coding of eDNA of a mitochondrial DNA sequence. Universal primers of fish mitochondria are used for PCR amplification of eDNA in a water body. Based on a high-throughput sequencing method, the problem that it is difficult to obtain effective species-specific primers when there are many related species in the water body can be solved effectively. Problems of difficult identification of related species DNA, large detection errors and so on can be avoided. The present disclosure can effectively and accurately detect freshwater fishes.
DESCRIPTION OF THE DRAWINGS Fig. 1 shows amplification results of 16s 200 fish universal primers on fish samples;
1. Zaccoplatypus; 2. Tachysurusfulvidraco, 3. Acheilognathusmacropterus; 4. Toxabramisswinhonis; 5. Culteralburnus; 6. Saurogobiodumerili, 7. Plagiognathopsmicrolepis;
8. Squaliduswolterstorffi, 9. Sinibramamacrops; 10. Hypophthalmichthysnobilis; 11. Cultermongolicus; 12. Hypophthalmichthysmolitrix; 13. Hemiculterleucisculus; 14. Sarcocheilichthyssinensis; 15. Pseudolaubucaengaulis; 16. Abbottinarivularis; 17.
Sarcocheilichthysnigripinnis; 18. Pseudobramasimoni; 19. Ctenogobiusgiurinus; 20. Sinipercachuatsi, 21. Carassiusauratus, 22. Chanodichthysdabryi; 23. Cyprinuscarpio; 24. Erythroculterdabryi, 25. Coilianasus; 26. Megalobramaskolkovii, 27. Xenocyprisargentea; 28.
Ctenopharyngodonidella; 29. Mylopharyngodonpiceus; and 30. negative control; and LU501782 Fig. 2 shows distribution of survey sampling points of fishes in Lake Gehu.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The technical solution of the present disclosure will be further described in detail with reference to the following drawings and specific embodiments. Embodiment 1 Verification of universal primers for fishes: universal primer 16s 200 successfully amplified DNA of 29 kinds of common fish. Muscle samples of 29 kinds of common fish are collected, and the DNA is extracted by using the animal genome DNA rapid extraction kit of Sangon Biotech (Shanghai) Co., Ltd. The extracted DNA is dissolved in TE buffer and stored at -20°C. The DNAs of 29 kinds of fish are subjected to PCR amplification by the synthesized primer 16s 200. Sample reaction system of 25 ul: 2.5 ul of 10xExTaq Buffer (Mg?*Plus) (20 mM), 2 ul of dNTP Mixture (2.5 mM for each), 1 ul of each of forward and reverse primers (10 umol/l), 0.2 ul of TaKaRa TagHS, 2 ul of DNA template, and 16.3 ul of ddH:O. Reaction conditions: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing for 40 s, extension at 72°C for 1 min, for 35 cycles; and extension for 10 min after 71°C. PCR products are detected by 1% gel electrophoresis. Amplification results of the primer 16s 200 on fish samples show that bright and clear bands appear at the same position in lanes 1-29 (see Fig. 1), indicating that the primer 16s 200 has a good amplification effect on all the 29 species of fish. Embodiment 2 27 species of fish distributed in Lake Gehu, Jiangsu Province, China were detected by primer 16s 200. We conducted field investigation and sampling in Lake Gehu, the second largest lake in southern Jiangsu Province of China, ranking only second to Taihu Lake. Lake Gehu (119°44'15"[119°52'56"E, 31°28'19"-31°43'04"N) is located in the southwest of Changzhou, Jiangsu Province. It is connected to the Yangtze River in the north, Taihu Lake in the east, Changdang Lake in the west and Dongjiu and Xijiu in Yixing City in the south. Main inflowing rivers into the lake include Biantan, Xiaxi, Huangli, Beigan and Zhonggan rivers, belonging to the river system of Taihu Lake. Lake Gehu is long and narrow in the south-north direction, and shaped like a long eggplant, with a length of 25 km and a width of 6.6 km, a water surface area of 164 km?, a water storage capacity of 1.74x10®m®, and an average water depth of 1.27 m. It is a typical shallow lake with various use functions such as drinking, irrigation, sightseeing and aquaculture. Lake Gehu is mainly of a subtropical monsoon climate, with mild climate and abundant rainfall all the year round, an annual average temperature of 15°C and annual average precipitation of about 1100 mm. At present, the data on fish diversity and fishery resources in Lake Gehu is insufficient. Therefore, we used the environmental DNA monitoring technology to investigate the fish composition distributed in 16 locations of Lake Gehu LU501782 (sampling points are shown in Fig. 2). (1) Water sample collection and treatment 16 sampling points were selected in Lake Gehu to collect water samples on a large scale.
Collection work was carried out in September 2017, May 2018 and November 2018. During sampling each time, the longitude and latitude were recorded by a GPS locator.
During sampling, clean plastic containers were used to collect 2 L of water samples (0.5 m deep) at each sampling point.
The collected water samples were put into a prepared foam box, which was filled with ice bags, transported back to the laboratory at a low temperature, and filtered by suction within 24 hours.
During suction filtration, a vacuum suction filter (AP-01 vacuum pump; AUTOS (IENCE)) was used to filter the water sample through a filter membrane with an aperture of 1.2 um, and we paid attention to prevent water sample pollution in the process of suction filtration.
The filtered filter membrane is immediately collected into a 2 mL of sterilized centrifuge tube and stored at -80°C. (2) DNA extraction After all samples were filtered by suction, the environmental DNA is extracted by using the kit QlAmp DNA Micro Kit produced by QIAGEN Company.
The specific steps are as follows: The filter membrane is cut with scissors, 180 pl of Buffer ATL and 20 ul of protein kinase K are added for shaking and mixing.
The mixture is incubated at 56°C until the mixture is completely lysed, with continuous shaking during incubation. 200 MI of BufferAL and 200 ul of ethanol are added for shaking and mixing; the above mixture is transferred to DNeasy Mini filter column in a 2 mL centrifuge tube with a pipette gun, and centrifuged at 6000xg (8000 rpm) for 1 min, and the filtrate and centrifuge tube are discarded.
The filter column is put into a new 2 mL collecting tube; 500 ul of Buffer AW1 is added; the mixture is centrifuged for 1 min at 26000xg; and the filtrate and collecting tube are discarded.
The filter column is transferred into a new 2 mL collecting tube; 500 pl of Buffer AW2 is added; the mixture is centrifuged at 20000xg (14000 rpm) for 3 min; and the filtrate and collecting tube are discarded.
The filter column to a new 1.5 mL centrifuge tube; 200 ul of Buffer AE is added to the center of the filter column membrane to elute DNA; and the mixture is incubated at a room temperature (15°C- 25°C) for 1 min, and centrifuged for 1 min at =6000xg.
The extracted DNA is dissolved in TE buffer and stored in a refrigerator at -20°C for later use. (3) DNA amplification The extracted environmental DNA samples are subjected to PCR amplification using the synthesized primer 16s 200. Sample reaction system of 25 ul: 2.5 pl of 10xExTaq Buffer (Mg**Plus) (20 mM), 2 ul of dNTP Mixture (2.5 mM for each), 1 ul of each of forward and reverse primers (10 umol/l), 0.2 ul of TaKaRa TaqHS, 2 ul of DNA template, and 16.3 ul of ddH2O.
Reaction conditions: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s,
annealing at 60.3°C for 40 s, extension at 72°C for 1 min, for 35 cycles; and extension for 10 LU501782 min after 71°C.
PCR products are detected by 1% gel electrophoresis, and the concentration is determined by NanoDrop™2000 spectrophotometer (Thermo FisherScience, Waltham, MA, USA). (4) lllumina Hiseq sequencing The PCR products amplified by the universal primer 16s 200 of fish are selected for sequencing.
Twenty-eight samples of eDNA are sequenced, and a total of 1,253,258 reads are generated.
The average length of sequenced fragments is 200 bp.
After optimization of original HiSeq data, 1,216,055 reads are reserved for subsequent analysis.
High-quality DNA sequences are grouped into 6,504 OTUS (Operational Taxonomic Units), and 3,398 OTUs can be annotated to known fish species.
In order to study the species composition of each sample, the EffectiveTags of all samples are clustered by OTUs with the Identity of 97%, among which 256 OTUs with 100% sequence identity are selected for further species annotation.
A total of 27 species of fish are identified from 28 eDNA samples (Table 3).
ANNAN A AAA AA AAA AR NANO AAA fable + Fish composition identified in cDNA samples LU501782 Lo Sep JT Mwy JR Raw 38088 N | 2 3 35.7 849 SIT TI VS ES IST OS IX 33 IS 153 18K 11 12 13 A ES ig TRÄNEN © vm a a a 2 vv ema aa OR a ee ve A A Ever Sealer iva RE CE 2 EE VAS A ee RS a ESS ES ES RS de a Ld 5
RS SEES ; ly su Sareea Ses Sees - -. “ x PE EE ES A ES aE a Ed a a a de ES ES ER Caras iamsg - 3 TRL Ev Bw bd EE RE RR Eh RE A EEE EE Onfiaverysdes : . 2 5 Lu 4 au © . ; Ca à V EN “ ~~ ~ ~ aN SRA a Ben EE dw Sao A a 3e 5
SENS Unfair . Co à Sa wa ~ ~ - a wa LE ES IEEE wa ~ ~ ~ oa ~ SF FUL a de a a a ES sh he Eb ad a a ES a he AR ES SE Rd bk € Fées SENTE . . ~ wa aa Na vv nn A ww kes ’
FEEDS sized Shrpoedoadeg SAT © TUT RE ER NS sh RE OS a RE CR ER ES SR SE ES ES SE
SEN Ss ARTE To ee EE SE ER ER A AE ES OHR
ET Sine ee PETE ve ma a ae Re EE ah aE a EEE vv vw hw Aw aw
SER SD FESTEN - x A x . . x N . . . - : - - - N x N : SE WOW A a a Te. PR WW AO a Er Eb de EE Snead EN ~ ~ ~ ~~ ~ ES a ~ X ~ ~ ~ + ne - A ce x X X > >. x. a Eh a £0 a hace SEES RR OO Ae ee be dE fe AO Creeley AS Co Ce Ce LA Lh a rE a Sd EE es a ee Ee SS Ee
AEE asser SNe vetoes eet S'ENSENSSRISN LL 4 4 Ce eee Cee eee » Fo Ee 4 SE 5 5 EE be BS A 6 A bE ER Ed A angel css
FERN TT PRE TT AST wm a ea a a vw wma aaa wT ew vw ww ewww Foxiseurects i “ - “ne ~ - - NN NS ae ~ ~ Ba a sea a Fae aa ies FX ÂFIETRERET ss
EN The above only describes preferred embodiments of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitution and improvements made within the spirit and principles of the present disclosure shall be contained within the protection scope of the present disclosure.
SEQUENCE LISTING LU501782 <110> Shanghai Ocean University <110> Xinyang Agriculture and Forestry University <120> PCR PRIMER, REAGENT OR KIT FOR IDENTIFYING OR ASSISTING IN
IDENTIFYING FRESHWATER FISH SPECIES AND IDENTIFICATION METHOD <130> SWPA-Fish Identification LU <150> CN202110572002.8 <151> 2022-05-25 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 45 <212> DNA <213> Artificial Sequence <400> 1 tgagtatggg agacagaaaa ggttcctttt gccacagaga cgggt 45 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <400> 2 tgagtatggg agacagaaaa ggttc 25 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 cttttgccac agagacgggt 20
Claims (7)
1. A PCR primer for identifying or assisting in identifying freshwater fish species, characterized in that the nucleotide sequence of the primer is SEQ ID NO.1 in the sequence listing.
2. The PCR primer according to claim 1, wherein the PCR primer is used for metabolic coding of eDNA of a fish mitochondrial DNA sequence.
3. A reagent or kit for identifying or assisting in identifying freshwater fish species, characterized in comprising the PCR primer of claim 1.
4. A method for identifying or assisting in identifying freshwater fish species by the PCR primer of claim 1 or the reagent or kit of claim 3, characterized in comprising: using the PCR primer of claim 1 for PCR amplification of a water sample to be detected, sequencing an amplified product, correcting or editing a sequence obtained by sequencing, submitting the sequence to a database for comparison, and identifying the fish species of the water sample according to a DNA sequence similarity.
5. The method according to claim 4, characterized in that in terms of 25 pl, an amplification system comprises the following components: 2.5 ul of 10 x ExTaq Buffer (Mg?*Plus) (20 mM), 2 ul of NTP Mixture (2.5 mM for each), 1 ul of each of forward and reverse primers (10 pmol/l), 0.2 pl of TaKaRa TagHS, 2 ul of DNA template, and 16.3 ul of ddH2O.
6. The method according to claim 4, characterized in that an amplification procedure is: pre- denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 60.3°C for 40 s, extension at 72°C for 1 min, for 35 cycles; and extension for 10 min after 71°C.
7. The method according to claim 6, characterized in that in the amplification procedure, an optimum annealing temperature of the PCR primer of claim 1 is 60.3°C.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110572002.8A CN113564262A (en) | 2021-05-25 | 2021-05-25 | PCR primer, reagent or kit for identifying or assisting in identifying freshwater fish species and identification method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| LU501782B1 true LU501782B1 (en) | 2022-10-03 |
Family
ID=78161569
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| LU501782A LU501782B1 (en) | 2021-05-25 | 2022-04-03 | Pcr primer, reagent or kit for identifying or assisting in identifying freshwater fish species and identification method |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN113564262A (en) |
| LU (1) | LU501782B1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115627295B (en) * | 2022-06-22 | 2023-10-27 | 深圳市华大海洋研究院 | Kit and whale species identification method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531879B (en) * | 2015-01-06 | 2017-02-22 | 上海海洋大学 | Environment DNA identification method for fish community structure researching |
| CN116656832A (en) * | 2019-04-26 | 2023-08-29 | 南京易基诺环保科技有限公司 | Freshwater fish mitochondria 12S universal macro barcode amplification primer and application method thereof |
-
2021
- 2021-05-25 CN CN202110572002.8A patent/CN113564262A/en active Pending
-
2022
- 2022-04-03 LU LU501782A patent/LU501782B1/en active IP Right Grant
Also Published As
| Publication number | Publication date |
|---|---|
| CN113564262A (en) | 2021-10-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xiong et al. | Zooplankton biodiversity monitoring in polluted freshwater ecosystems: A technical review | |
| Sigsgaard et al. | Seawater environmental DNA reflects seasonality of a coastal fish community | |
| Cowart et al. | Metagenomic sequencing of environmental DNA reveals marine faunal assemblages from the West Antarctic Peninsula | |
| Zou et al. | eDNA metabarcoding as a promising conservation tool for monitoring fish diversity in a coastal wetland of the Pearl River Estuary compared to bottom trawling | |
| Deheyn et al. | Evidence for enhanced bioavailability of trace elements in the marine ecosystem of Deception Island, a volcano in Antarctica | |
| Sahu et al. | Environmental DNA (eDNA): Powerful technique for biodiversity conservation | |
| Harper et al. | Finding crush: environmental DNA analysis as a tool for tracking the green sea turtle Chelonia mydas in a marine estuary | |
| Massana et al. | Growth of uncultured heterotrophic flagellates in unamended seawater incubations | |
| LU501782B1 (en) | Pcr primer, reagent or kit for identifying or assisting in identifying freshwater fish species and identification method | |
| EP3022317B1 (en) | Microarray slide and method for detecting toxic algae | |
| Lehman et al. | Environmental DNA evidence of the Critically Endangered smalltooth sawfish, Pristis pectinata, in historically occupied US waters | |
| KR20160000035A (en) | Identifying Method of microalgae Nitzschia improvisa in Southern Sea of Korea, Polynucleotide Probe, DNA Chip and Kit for Identifying The Same | |
| KR101151757B1 (en) | Identifying Method of jellyfish in Southern Sea of Korea, Polynucleotide Probe, DNA Chip and Kit for Identifying The Same | |
| Von der Heyden et al. | Species‐specific genetic markers for identification of early life‐history stages of Cape hakes, Merluccius capensis and Merluccius paradoxus in the southern Benguela Current | |
| Wang et al. | Haplosporidium nelsoni and H. costale in the Pacific oyster Crassostrea gigas from China’s coasts | |
| Cheng et al. | Sex Identification of the Black-faced Spoonbill(Platalea minor) | |
| Rukminasari et al. | Species assemblages and distribution of Dinoflagellate cysts from three estuaries sediment’s of Makassar Strait, Eastern Indonesia | |
| Guy-Haim et al. | A new record of the rapidly spreading calanoid copepod Pseudodiaptomus marinus (Sato, 1913) in the Levantine Sea using multi-marker metabarcoding. | |
| CN111926088B (en) | Primer group, kit and method for detecting salmon population | |
| Yue et al. | Molecular monitoring plankton community during jellyfish blooms in the water near the Liaoning Hongyan River Nuclear Power Plant | |
| Chen et al. | An optional low-cost method of extracting environmental DNA of macro-organisms from filter membranes in large scale eDNA surveys | |
| Hogg et al. | Biodiversity genomics: monitoring restoration efforts using DNA barcoding and environmental DNA | |
| CN113005208B (en) | Universal macro-barcode amplification primer for mollusks and application method thereof | |
| CN113512593B (en) | Mussel environment DNA macro-barcode primer, identification method and application | |
| Hartanto et al. | Environmental suitability and detection of cnidarian invasive species at Dumai and Kuala Tanjung Port using eDNA |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FG | Patent granted |
Effective date: 20221003 |