Primer group and kit for detecting invading sunfish species based on environmental DNA (Deoxyribose Nucleic Acid)

Abstract

The invention belongs to the technical field of biology, and particularly discloses a primer combination, a kit and a method for detecting an invasive species sunfish in water by utilizing multiple PCR (Polymerase Chain Reaction). According to the invention, a primer design method is innovated, a dominant sunfish multiple PCR primer combination is screened out, and the sunfish DNA can be detected under the background of various types of fishes in combination with an optimized PCR reaction system and conditions, so that the problem of environmental eDNA detection is solved, multiple PCR primers can be combined with environmental eDNA detection, and the diffusion trend of the sunfish can be monitored more accurately. The detection method can effectively save time and cost, has high sensitivity, strong specificity, high efficiency and low professional requirements, has a good detection rate when the concentration of a target species is low, and can directly obtain a result through gel electrophoresis.

Description

Technical field:

[0001] The invention relates to the field of biotechnology, in particular to a group of primers for detecting invasive species of the family Sunfish and applications thereof. Background technology:

[0002] The sunfish family belongs to the phylum Vertebrata, order Perciformes, and is mainly freshwater fish. Most of the sunfish are native to the Americas, mainly distributed in the central plains of North America, including northeastern Mexico and southeastern Canada.

[0003] Green sunfish (Lepomis cyanellus) is one of the most common sunfish in China. It belongs to the Perciformes, Porcoidei, Cehtrachidae, and Lepomis. It was originally distributed in streams and lakes from the Great Lakes of North America to Mexico. It inhabits still water in slow-moving streams, lakes, and ponds. It often appears near plants and is carnivorous. Juveniles feed on aquatic insects and small crustaceans. It is a famous sport fish that can be eaten or viewed. It is also an important farmed fish.

[0004] The bluegill sunfish (Lepomis macrochirus) belongs to the Perciformes, Porcoidei, Cehtrachidae, and Lepomis. It was originally distributed in streams and lakes from the Great Lakes of the St. Lawrence River in North America and the Mississippi River basin to northern Mexico. The bluegill sunfish is brown with light blue stripes, and has many vertical stripes on its body, separated by light lines. The edge of the gill cover is light blue, hence the name. The bluegill sunfish likes to live in lakes, ponds, reservoirs, and slow-moving streams. It is active at dusk and dawn. It is carnivorous, and the adult fish feeds on snails, crayfish, insects, worms, and fish. The juvenile fish preys on crustaceans, insects, and worms. It is an important sport fish, and has commercial value as an ornamental fish and food fish.

[0005] Largemouth bass (Micropterus salmoides), commonly known as California bass and black bass, belongs to the Perciformes, Porcoidei, Cehtrachidae, Micropterus genus, native to North America, and is a worldwide sport fish. According to the geographical distribution and morphological differences of the origin, largemouth bass is divided into two subspecies: one is the northern subspecies (M. salmoides salmoides) distributed in the central and eastern United States, northeastern Mexico and southeastern Canada; the other is the Florida subspecies (M. salmoides floridanus) distributed in southern Florida. Taiwan Province of my country introduced this fish from abroad in the late 1970s, and artificial breeding was successful in 1983. In the same year, it was introduced to Guangdong Province. After years of breeding development, it has become one of the important freshwater aquaculture varieties in China.

[0006] Sunfish are mostly introduced into my country as ornamental and sport fish, and have been promoted for breeding throughout the country. However, due to their wide adaptability, omnivorous diet, high population yield, and natural reproduction, they have caused a considerable impact on the local native ecosystem. In China, reports of invasions of different sunfish are common, such as the green sunfish, which has been reported to have invaded the Pihe River Basin in the Dabie Mountains of Anhui Province and the Jialing River in Sichuan Province. However, due to the small number of individuals, it is not easy to detect the early invasion of alien species. Environmental DNA refers to DNA directly obtained from environmental samples, including DNA released by animals, plants, and microorganisms in the air, soil, water, and other environments. Environmental DNA technology can detect cryptic species in water bodies through species-specific molecular markers. Therefore, it is of great significance to establish a method for quickly and accurately identifying early invasions of sunfish based on environmental DNA. Summary of the invention:

[0007] The technical problem to be solved by the present invention is to provide a primer set, a kit and an application thereof capable of detecting invasive species of the family Sunfish, so as to realize early invasion detection of species of the family Sunfish based on environmental DNA.

[0008] The technical solution of the present invention to solve the above problems is as follows:

[0009] In a first aspect, a primer set for detecting invasive species of the family Sunfish in an environment is provided. The primer set includes three sets, and the sequences of the three primer sets are shown in SEQ ID NO.1-6.

[0010] Furthermore, the three primer sets and their corresponding amplification intervals are:

[0011] The sequences of the first primer set are shown in SEQ ID NO. 1-2, and are used to amplify the D-loop region on mitochondria;

[0012] The sequences of the second primer set are shown in SEQ ID NO. 3-4, and are used to amplify the D-loop region on mitochondria;

[0013] The sequences of the third primer set are shown in SEQ ID NO. 5-6, and are used to amplify the D-loop region on mitochondria.

[0014] Among them, SEQ ID NO 1, SEQ ID NO 3, and SEQ ID NO 5 are forward primers (F) in each primer set, and SEQ ID NO 2, SEQ ID NO 4, and SEQ ID NO 6 are reverse primers (R) in each primer set.

[0015] Furthermore, as a preference, each primer set may add a molecular tag sequence at the 5' end, wherein the unique molecular tag is a random sequence of 6-9 bp, preferably a random sequence of 8 bp.

[0016] In a second aspect, a kit for detecting invasive sunfish species in an environment is provided, the kit comprising the above-mentioned primer set.

[0017] Further, the kit comprises a DNA polymerase and a buffer thereof. Preferably, the DNA polymerase may be Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase or Klenow fragment.

[0018] Furthermore, the kit comprises a positive control, and preferably, the DNA positive control is an amplification sequence corresponding to the primer set.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The primer set or kit for detecting invasive species provided by the present invention can simultaneously detect three molecular markers located on the mitochondrial genome of invasive sunfish species, can detect invasive sunfish species in the presence of background DNA of multiple fish species, and has the characteristics of high specificity, high accuracy, high sensitivity, etc. Description of the drawings:

[0021] Figure 1: The results of the specificity test of a single primer on common freshwater fishes of closely related species of the target species in Example 3. In the figure, Marker: DNA Marker; Blank (negative control): DNA is replaced with sterile enzyme-free water, and any pair of primers SEQ ID NO1 / 2, SEQ ID NO 3 / 4, SEQ ID NO 5 / 6 is used; SEQ ID NO 1 / 2: primer pair SEQ ID NO 1 / 2 is used, and green sunfish DNA is used as shown in the figure. And so on. Positive control: using fish universal primer Mifish, sunfish DNA.

[0022] Figure 2 : The specificity test result diagram of a single primer in Example 3 for common freshwater fish of closely related species of the target species. The specificity test includes closely related species of sunfish and 20 common freshwater fish. In the figure, Marker: DNA Marker; Blank (negative control): DNA is replaced with sterile enzyme-free water, and any pair of primers SEQ ID NO 1 / 2, SEQ ID NO 3 / 4, SEQ ID NO 5 / 6 are used; Close: DNA of closely related species, using primer SEQ ID NO 1 / 2; Fresh: DNA of 20 common freshwater fish, using primer SEQ ID NO 1 / 2; As shown in the figure, and so on. Positive control: using fish universal primer Mifish, sunfish DNA.

[0023] Figure 3 : The amplification effect diagram of the primer combination in Example 3 on the target species bluegill sunfish, green sunfish and largemouth bass. In the figure, Marker: DNA Marker; Blank (negative control): DNA is replaced with sterile enzyme-free water, and the primer combination of SEQ ID NO 1 / 2, SEQ ID NO 3 / 4, and SEQ ID NO 5 / 6 is used; SEQ ID NO 1 / 2 3 / 4 5 / 6: primer combination SEQ ID NO 1 / 2, SEQ ID NO 3 / 4, SEQ ID NO 5 / 6 is used; SEQ ID NO 1 / 2: primer SEQ ID NO1 / 2 is used. And so on as positive control. The DNA used is bluegill sunfish tissue-like DNA, green sunfish tissue-like DNA, and largemouth bass tissue-like DNA.

[0024] Figure 4: The effect of primers in Example 3 on single and combined amplification of sunfish environmental DNA from natural water species. In the figure, Marker: DNA Marker; Blank (negative control): DNA is replaced with sterile enzyme-free water, and primers SEQ ID NO 1 / 2, SEQ ID NO 3 / 4, and SEQ ID NO 5 / 6 are used; 1×: natural water stock solution; 10×: natural water is diluted 10 times, and the DNA used is sunfish natural water DNA; SEQ ID NO 1 / 2: primers SEQ ID NO 1 / 2 are used. And so on as positive controls.

[0025] Figure 5 : Electrophoresis results of the sensitivity test of each primer pair in Example 4. In the figure, Marker: DNAMarker; Blank (negative control): DNA is replaced with sterile enzyme-free water, and any pair of primers SEQ ID NO 1 / 2, SEQ ID NO 3 / 4, SEQ ID NO 5 / 6 is used. 1: Sunfish tissue sample stock solution DNA Sunfish 5525.52ng / ml; 2: DNA Sunfish 1105.1ng / ml; 3: DNA Sunfish 221.02ng / ml; 4: DNA Sunfish 44.21ng / ml; 5: DNA Sunfish 8.85ng / ml; 6: DNA Sunfish 1.77ng / ml; Positive control: using fish universal primers Mifish, sunfish tissue sample stock solution DNA. Specific implementation method:

[0026] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0027] If the specific technology or conditions are not specified in the examples, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased through regular channels. The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0028] Example 1: Design of primer set

[0029] The mitochondrial genome sequences of bluegill sunfish (Lepomis macrochirus, NC_015984.2), green sunfish (Lepomis cyanellus, NC_020359.1), and largemouth bass (Micropterus salmoides, NC_008106.1) published by the National Center for Biotechnology Information Gene Bank (NCBI Gene Bank) were used as reference sequences for the target species.

[0030] The designed primer set should meet the following requirements at the same time:

[0031] 1) Other sunfish species other than the target species cannot be expanded simultaneously;

[0032] 2) The amplified target fragment sequence can distinguish different target sunfish.

[0033] The designed primer set should also meet the following requirements:

[0034] 1) The target species and all background species differ by more than 3 bp in the primer binding region and meet one of the following conditions:

[0035] The last 1 bp at the 3' end is different;

[0036] The last 4 bases at the 3' end differ by 2 bp;

[0037] The last 6 bases at the 3' end differ by 3 bp.

[0038] 2) Amplified fragment size, 100bp-150bp;

[0039] 3) Primer size, 22 bp-29 bp;

[0040] 4) Annealing temperature, 53-57°C;

[0041] 5) GC content, 20%-80%;

[0042] 6) Hairpin, <40;

[0043] 7)polyX<7.

[0044] Based on the above principles, the present invention designs three sets of primers, as shown in the sequence table. The present invention has no special restrictions on the source of each primer of the designed primer set, and the primer source known in the art can be used. In the embodiment of the present invention, the primer set is commissioned to be synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0045] Example 2: Specificity and sensitivity of primer sets

[0046] In order to verify the amplification effect of the primer set of the present invention on the target species, this example used three primer sets to amplify the DNA of the target species. The results are shown in the attached Figure 1 The three primer sets described in the present invention all have good amplification effects on target species.

[0047] Furthermore, in order to verify the specificity of the primer set of the present invention, this example selected 20 common freshwater fish and 4 closely related species of tilapia as background species. The 20 common freshwater fish species include: Channa argus, Paramisgurnus dabryanus, Hypophthalmichthys molitrix, Xenocypris argentea, Hypophthalmichthys nobilis, Culteralburnus, Rhodeus ocellatus, Cyprinus carpio, Paracanthobrama guichenoti, Tachysurus fulvidraco, Saurogobiodumerili, Pseudorasbora parva, Abbottina rivularis, Abbottina rivularis, (Hemibarbus maculatus), yellowtail (Micropercops swinhonis), crucian carp (Carassius auratus), grass carp (Mylopharyngodon piceus), (Hemiculterleucisculus) and black carp (Mylopharyngodon piceus). The four closely related species are freshwater grouper (Cichlasoma managuense), Nile tilapia (Oreochromis niloticus), Aureus tilapia (Oreochromis aureus) and Coptodon zillii. In this example, the three primer sets were used to amplify the DNA of all background species. Figure 2 As shown, none of the three pairs of primers amplified the closely related species of sunfish and 20 common freshwater fish species. It was preliminarily judged that they can be used as primers for amplifying sunfish DNA.

[0048] Furthermore, in order to achieve multi-site detection, this example combined the primers and tested tissue-like DNA and environmental DNA from natural water bodies. The test results are shown in the attached Figure 3 and attached Figure 4 The results are shown in Figure 3 As shown in the figure, it can be seen that when three pairs of primers are used to amplify three sunfish tissue-like DNAs, only two clear bands can be seen because the lengths of the amplified products of SEQ ID NO 1 / 2 and SEQ ID NO 3 / 4 primers are similar. Figure 4 As shown, when three pairs of primers were used to amplify the natural water sample of sunfish, two bands were seen. The amplification efficiency of the natural water environmental DNA was better than the amplification effect of the natural water environmental DNA after diluting it 10 times.

[0049] Example 3: Preparation of the kit

[0050] On the basis of Example 1 and Example 2, Example 3 simultaneously selects the above three primer groups to prepare a kit for detecting early invasion of sunfish in the environment to illustrate the kit and its preparation method.

[0051] The primer set can realize multi-site simultaneous detection, and can achieve the purpose of detecting early-stage invasive sunfish in the environment with high accuracy and high throughput.

[0052] Prepare PCR amplification reaction reagents, mainly including primer sets, enzymes, dNTPs, PCR buffer (1×TE buffer: prepared from Tris and EDTA), and PCR additives, including but not limited to betaine, fetal bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), tetramethylammonium chloride (TMAC), formamide and trehalose, and assemble into a kit according to specifications.

[0053] The above steps are used to prepare PCR amplification reaction solution, quality control product (target amplified fragment DNA), and PCR water to assemble into a detection kit for early invasive sunfish in the environment.

[0054] Example 4: Detection of invasive fish species of the family Sunfish based on environmental DNA.

[0055] This embodiment provides a method for detecting an invasive species, sunfish, in natural water bodies using a multi-site method, comprising the following steps:

[0056] S1. Water sample extraction:

[0057] Water samples from natural water bodies where invasive fish of the family Sunfish exist were filtered using a 0.45 μm mixed cellulose membrane to enrich environmental DNA, and the filter membrane was stored in a centrifuge tube containing 1200 μL of sterile CTAB storage solution to immerse the filter membrane.

[0058] S2. eDNA water sample extraction:

[0059] Oscillate the centrifuge tube vertically at low speed for 5 minutes and place it in a 60℃ constant temperature water bath for 30 minutes. After taking it out, add 1200μL of chloroform: isoamyl alcohol (24:1) to each tube and oscillate vertically at low speed for 7 minutes to allow the solution to react fully. Centrifuge at 7000rpm for 15 minutes at room temperature to separate the aqueous phase and the organic phase. Pipette 1000μL of the aqueous phase into a 2ml centrifuge tube, add 500μL of ice-cold isopropanol and 250μL of 5MNaCl solution respectively, and then place it in a -20℃ refrigerator for 1 hour. Centrifuge at 12000rpm for 15 minutes at room temperature and discard the supernatant. Add 150μL of 70% alcohol to clean the inner wall of the centrifuge tube, centrifuge at 15000rpm for 5 minutes at room temperature, discard the alcohol, and repeat this step twice. After rinsing, open the centrifuge tube cap and dry it at room temperature for 10 minutes. Finally, add 50 μL of TE solution to each centrifuge tube and heat in a 55°C water bath for 10 min to obtain water sample eDNA, which was then stored in a -20°C refrigerator for later use.

[0060] S3. Multiplex PCR amplification:

[0061] The multiplex PCR system was prepared using the amplification kit in Example 3: 5 μL of PCR amplification reaction solution, 3 μL of DNA template, and sterile enzyme-free water to make up to 10 μL. Each component was mixed by oscillation and placed in a PCR instrument.

[0062] The PCR amplification procedure is as follows:

[0063] Pre-denaturation at 95°C for 5 min;

[0064] Denaturation at 95°C for 30 s, annealing at 56.6°C for 90 s, extension at 72°C for 30 s, and the reaction cycle was repeated 40 times;

[0065] Extension at 68°C for 10 min;

[0066] Store at 12℃.

[0067] S4. Electrophoresis detection:

[0068] Prepare a 1.5% agarose gel. After the gel solidifies, take 1 μL of PCR product, mix it with 1 μL of loading buffer, and then apply it to the electrophoresis instrument at 110V and 400mA for 40 minutes. Observe the bright bands in the gel imager to preliminarily determine the presence of the target species, sunfish. Figure 4As shown in the figure, using three pairs of primers to amplify the sunfish natural water sample, two bands can be seen, and the amplification efficiency of the natural water stock solution is better than that of the natural water 10-fold dilution. Since the bp numbers of Centra1 and Centra2 primers are similar, they cannot be distinguished by gel images. It can be seen that the amplification efficiency of tissue samples is greater than that of natural water.

[0069] S5. Sequencing verification: The multiplex PCR amplification products were recovered and purified using a DNA purification recovery kit, and the purified samples were sent to Biotechnology Co., Ltd. for sequencing. The sequences obtained by sequencing were subjected to BLAST comparison analysis.

Claims

1. A primer set for detecting invasive species of sunfish, bluegill sunfish, green sunfish and largemouth bass in water based on environmental DNA, characterized in that: The primer set includes 3 groups, and the sequences of the 3 primer groups are shown as SEQ ID NO.1-6.

2. The primer set according to claim 1, characterized in that: The three sets of primers and their corresponding regions are: The sequences of the first primer set are shown in SEQ ID NO. 1-2, and are used to amplify the D-loop region in the mitochondria of the target species; The sequences of the second primer set are shown in SEQ ID NO. 3-4, and are used to amplify the D-loop region in the mitochondria of the target species; The sequences of the third primer set are shown in SEQ ID NO. 5-6, and are used to amplify the D-loop region in the mitochondria of the target species.

3. According to claim 1, it is characterized in that: The primer set includes any one of the base sequences SEQ ID NO. 1 to 6.

4. According to claim 1, it is characterized in that: The primer set includes any two sets of base sequences SEQ ID NO.1-6.

5. According to claim 1, it is characterized in that: The primer set includes any three sets of base sequences SEQ ID NO. 1-6.

6. According to claim 1, it is characterized in that: All three primer sets were able to detect bluegill sunfish, green sunfish, and largemouth bass.

7. According to claim 1, it is characterized in that: A molecular tag sequence may be added to the 5' end of the primer, wherein the unique molecular tag is a random sequence of 6-9 bp, preferably a random sequence of 8 bp.

8. The primer set according to any of claim 1 is used in any of the following applications: (1) Identify or assist in the identification of bluegill sunfish; (2) preparing products for identifying or assisting in the identification of bluegill sunfish; (3) Identify or assist in the identification of green sunfish; (4) preparing products for identifying or assisting in the identification of green sunfish; (5) Identify or assist in the identification of largemouth bass; (6) Preparing products for identifying or assisting in the identification of largemouth bass.

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