Primer group and kit for detecting aquatic invasive species tilapia mossambica 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 invasive species tilapia mossambica in water by utilizing multiple PCR (Polymerase Chain Reaction). According to the invention, a primer design method is innovated, a dominant tilapia multiple PCR primer combination is screened out, and by combining with an optimized PCR reaction system and conditions, tilapia DNA can be detected under the background of various types of Sagittaria trifolia, the problem of environmental eDNA detection is solved, multiple PCR primers can be combined with environmental DNA detection, and the trend of tilapia can be monitored more accurately. The detection method can effectively save time and cost, is high in sensitivity, specificity and efficiency and low in professional requirement, the detection rate is better when the concentration of a target species is low, and a result can be directly obtained through gel electrophoresis.

Description

Technical Field:

[0001] The present invention relates to the field of biotechnology, and particularly relates to a set of primers for detecting the invasion of tilapia and their applications. Background Art:

[0002] Tilapia is a type of freshwater fish belonging to the order Perciformes, family Cichlidae, and genus Oreochromis, and is an invasive species in China. A series of characteristics suitable for farming, such as fast growth and reproduction rates and strong environmental adaptability, have also promoted its spread and invasion in natural waters, making it a "typical invasive species". Currently, most tropical and subtropical regions of the world are threatened by tilapia invasion, and tilapia has also become one of the most widely distributed alien fish species globally. Natural populations of tilapia have been found in the Pearl River Basin, Hainan, Guangxi, and various waters in Fujian in China. Moreover, the distribution range of tilapia in China is gradually expanding northward, and there have been reports of tilapia invasion in water bodies such as the Qiantang River and Qiandao Lake. However, due to the small number of individuals, it is not easy to detect the early stage of alien species invasion. Environmental DNA refers to DNA directly obtained from environmental samples, including DNA released by animals, plants, and microorganisms into the environment such as air, soil, and water bodies. 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 the early invasion of tilapia based on environmental DNA. Summary of the Invention:

[0003] The technical problem to be solved by the present invention is to provide a set of primer sets, kits, and their applications for detecting the invasive species tilapia. The purpose is to achieve the discovery of the early invasion of tilapia based on environmental DNA.

[0004] The technical solution of the present invention to solve the above problems is as follows:

[0005] In the first aspect, a set of primer sets for detecting tilapia DNA in the environment is provided. The primer sets include 4 groups, and the sequences of the 4 groups of primer sets are shown in SEQ ID NO.1-8.

[0006] Furthermore, the 4 groups of primer sets and their corresponding amplification regions are respectively:

[0007] The sequence of the first group of primer sets is shown in SEQ ID NO.1-2, and is used for amplifying the 16S ribosomal RNA region on the mitochondrion;

[0008] The sequence of the second group of primer sets is shown in SEQ ID NO.3-4, and is used for amplifying the ND1-tRNA-Ile region on the mitochondrion;

[0009] The sequence of the third group of primer sets is shown in SEQ ID NO.5-6, and is used for amplifying the COI region on the mitochondrion;

[0010] The sequences of the fourth primer set are shown in SEQ ID NO. 7-8 and are used to amplify the D-loop region on the mitochondria.

[0011] Among them, SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.7 are the forward primers (F) in each primer set, and SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8 are the reverse primers (R) in each primer set.

[0012] Furthermore, preferably, a molecular tag sequence can be added to the 5' end of each primer set, and the unique molecular tag is a random sequence of 6-9 bp, preferably an 8-bp random sequence.

[0013] In a second aspect, a kit for detecting the invasive species tilapia in the environment is provided, and the kit contains the above-mentioned primer set.

[0014] Furthermore, the kit contains a DNA polymerase and its buffer. Preferably, the DNA polymerase can 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 the Klenow fragment.

[0015] Furthermore, the kit contains a positive control. Preferably, the DNA positive control is the amplification sequence corresponding to the primer set.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] The primer set or kit for detecting invasive species provided by the present invention can simultaneously detect 4 molecular markers located on the mitochondrial genome of tilapia, and can detect tilapia DNA in the presence of various fish background DNAs, with the characteristics of strong specificity, high accuracy, and high sensitivity. Description of the drawings:

[0018] Figure 1 : Electrophoresis results of amplifying the DNA of the target species with a single primer pair in Example 3. In the figure, Marker: DNA Marker; Blank (negative control): DNA is replaced with sterile and enzyme-free water, using any one of the four primer sets; 1: Using primer set SEQ ID NO.1, SEQ ID NO.2, using tilapia DNA as shown. And so on. Mifish (positive control): Using the general fish primer Mifish, tilapia DNA.

[0019] Figure 2 : Amplification specificity result graph of a single primer pair for the target species, its related species, and common freshwater fish in Example 3. The specificity test includes related species of tilapia and 20 common freshwater fish. In the graph, Marker: DNA Marker; Blank (negative control): DNA is replaced with sterile and enzyme-free water, using any one of primer pairs SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8; Near: DNA of related species, using primer Tila1; Fresh: DNA of 20 common freshwater fish, using primer Tila1; As shown in the figure, and so on. Mifish (positive control): Using the universal fish primer Mifish and tilapia DNA.

[0020] Figure 3 : Test result graph of primer combination for amplifying DNA of the target species in Example 3. In the graph, Marker: DNA Marker; 1246: Using primer combination SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8. 1: Using primer SEQ ID NO.1-2. And so on for positive controls. The DNAs used are tissue sample DNA of Oreochromis niloticus, Oreochromis aureus, and Oreochromis mossambicus respectively.

[0021] Figure 4 : Electrophoresis result graph of the optimal annealing temperature test of primer combination in Example 3. The left side is the amplification effect graph of the mixed primer under gradient annealing temperatures, and the right side is the positive control result of the individual amplification of four primer pairs. In the graph, Marker: DNA Marker; 1 2 3 4 5 6 represent the amplification efficiency of multiplex PCR under different temperature gradients, using environmental DNA for amplification. The result shows that the best effect is achieved at 56.6 °C.

[0022] Figure 5 : Electrophoresis result graph of the test of primer combination for environmental DNA in Example 3.. In the graph, Marker: DNA Marker; 1×: Natural water stock solution; 10×: Natural water diluted 10 times; Tila: Using primer combination SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8.

[0023] Figure 6: Electrophoresis result diagram of the sensitivity test of single pair of primers in Example 4. In the figure, Marker: DNA Marker; Blank (negative control): DNA is replaced with sterile and enzyme-free water, and any one pair of primers SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8 is used. 1: Original solution DNA of tilapia tissue sample, tilapia 16061.25 ng / ml; 2: DNA tilapia 3212.25 ng / ml; 3: DNA tilapia 642.45 ng / ml; 4: DNA tilapia 128.49 ng / ml; 5: DNA tilapia 25.698 ng / ml; 6: DNA tilapia 5.1396 ng / ml; Mifish (positive control): Use the general fish primer Mifish and the original solution DNA of tilapia tissue sample. As shown in the figure, for 1, 2, 3, 4, 5, 6, primers SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8 are used respectively. Detailed implementation manners:

[0024] Reference to the embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, features described or illustrated as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0025] For those technical or conditions not specified in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be purchased through regular channels. The present invention will be further described below in conjunction with the accompanying drawings and specific examples.

[0026] Example 1: Design of primer sets

[0027] Based on the mitochondrial genome sequences of Nile tilapia (Oreochromis niloticus, NC_013663.1), Zill's tilapia (Coptodon zillii, NC_026110.1), and blue tilapia (Oreochromis aureus, NC_013750.1) publicly available in the National Center for Biotechnology Information Gene Bank (NCBI Gene Bank) as the reference sequences of the target species.

[0028] The designed primer sets should simultaneously meet the following requirements:

[0029] 1) It is not possible to simultaneously amplify other cichlid fish species except the target species;

[0030] 2) The amplified target fragment sequence can distinguish different target tilapia species.

[0031] The designed primer sets should also meet the following requirements:

[0032] 1) There are more than 3 bp differences between the target species and all background species in the primer binding region, and one of the following situations is satisfied:

[0033] The last 1 bp at the 3' end is different;

[0034] 2 bp out of the last 4 bases at the 3' end are different;

[0035] 3 bp out of the last 6 bases at the 3' end are different.

[0036] 2) The size of the amplified fragment is 100 bp - 150 bp;

[0037] 3) The size of the primer is 22 bp - 29 bp;

[0038] 4) The annealing temperature is 53 - 57 °C;

[0039] 5) The GC content is 20% - 80%;

[0040] 6) Hairpin < 40;

[0041] 7) polyX < 7.

[0042] Based on the above principles, the present invention has designed a total of four primer sets as shown in the sequence listing. There are no special restrictions on the sources of the primers of the designed primer sets in the present invention, and the well-known primer sources in the art can be used. In the examples of the present invention, the primer sets were entrusted to Genewiz (Suzhou) Inc. for synthesis.

[0043] Example 2: Specificity and sensitivity of primer sets

[0044] To verify the amplification effect of the primer sets described in the present invention on the target species, in this example, four primer sets were respectively used to amplify the DNA of the target species, and the results are shown in the appendix Figure 1 The four primer sets described in the present invention all have good amplification effects on the target species.

[0045] Further, to verify the specificity of the primer sets of the present invention, 19 common freshwater fish species and 4 tilapia-related species were selected as background species in this example. The 19 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, Micropercops swinhonis, Carassius auratus, Mylopharyngodon piceus, (Hemiculter leucisculus) and Mylopharyngodon piceus. The four tilapia-related species are Cichlasoma managuense, Micropterussalmoides, Lepomis auritus, and Lepomis macrochirus. In this example, the four primer sets were used to amplify the DNA of all background species. The results are shown in the appendix Figure 2 as follows. None of the four primer sets could amplify the DNA of the 4 tilapia-related species and 20 common freshwater fish species.

[0046] Further, to achieve multi-locus detection, the primers were combined in this example and tested on tissue sample DNA and environmental DNA from the aquaculture water. The test results are shown in appendix Figure 3 and appendix Figure 4 respectively. As shown in Figure 3 , when 4 pairs of primers were used to amplify the DNA of tissue samples of 3 tilapia species, 3 clear bands could be seen. Since the amplified product lengths of the primers SEQ ID NO.1-2 and SEQ ID NO.7-8 are similar, they could not be distinguished by the gel image. As shown in Figure 4As shown, 4 pairs of primers were used to amplify the water samples from tilapia farms, and 3 bands could be seen. The amplification efficiency was the highest at 56.6°C. Since the primer bp numbers of SEQ ID NO.1-2 and SEQ ID NO.7-8 were similar, they could not be distinguished by the gel image.

[0047] Example 3: Preparation of the kit

[0048] Based on Example 1 and Example 2, Example 3 simultaneously selected the above four primer sets to prepare a kit for detecting early invasive tilapia in the environment to illustrate the kit and its preparation method.

[0049] Through the primer set, multi-locus simultaneous detection can be achieved, and the purpose of accurately and highly detecting early invasive tilapia in the environment can be achieved.

[0050] Prepare the PCR amplification reaction reagents, mainly including the primer set, enzyme, dNTPs, PCR buffer (1×TE buffer: composed of Tris and EDTA), and PCR additives, including but not limited to betaine, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), tetramethylammonium chloride (TMAC), formamide, and trehalose, etc., and assemble them into a kit according to the specifications.

[0051] Assemble the PCR amplification reaction solution, quality control product (target amplified fragment DNA), and PCR water prepared in the above steps into a detection kit for early invasive tilapia in the environment.

[0052] Example 4: Detection of invasive tilapia based on environmental DNA.

[0053] This example provides a method for detecting invasive species tilapia in natural waters using a multi-locus method, including the following steps:

[0054] S1. Water sample extraction:

[0055] Use a 0.45μm mixed cellulose membrane to filter the water samples from natural waters where tilapia exists, enrich the environmental DNA, and store the filter membrane in a centrifuge tube containing 1200μL of sterilized CTAB preservation solution, submerging the filter membrane.

[0056] S2. eDNA water sample extraction:

[0057] Vortex the centrifuge tube vertically at low speed for 5 minutes, and place it in a constant temperature water bath at 60 °C for 30 minutes. After taking it out, add 1200 μL of chloroform:isoamyl alcohol (24:1) to each tube, vortex vertically at low speed for 7 minutes to allow the solution to react fully. Centrifuge at 7000 rpm for 15 minutes at room temperature to separate the aqueous phase from the organic phase. Pipette 1000 μL of the aqueous phase into a 2 mL centrifuge tube, add 500 μL of ice-cold isopropanol and 250 μL of 5 M NaCl solution respectively, and then place it in a -20 °C refrigerator for 1 hour. Centrifuge at 12000 rpm for 15 minutes at room temperature, and discard the supernatant. Add 150 μL of 70% alcohol to wash the inner wall of the centrifuge tube, centrifuge at 15000 rpm for 5 minutes at room temperature, and discard the alcohol. Repeat this step twice. After rinsing thoroughly, open the centrifuge tube lid at room temperature and let it dry for 10 minutes. Finally, add 50 μL of TE solution to each centrifuge tube, heat it in a 55 °C water bath for 10 minutes to obtain the water sample eDNA, and store it in a -20 °C refrigerator for later use.

[0058] S3. Multiplex PCR amplification:

[0059] Configure the multiplex PCR system using the amplification kit in Example 3: Among them, 5 μL of PCR amplification reaction solution, 3 μL of DNA template, and sterile and enzyme-free water are added to make up to 10 μL. Mix each component by oscillation and place it in a PCR instrument.

[0060] The PCR amplification program is as follows:

[0061] Pre-denaturation at 95 °C for 5 minutes;

[0062] Denaturation at 95 °C for 30 s, annealing at 56.6 °C for 90 s, extension at 72 °C for 30 s, and cycle reaction for 40 times;

[0063] Extension at 68 °C for 10 minutes;

[0064] Store at 12 °C.

[0065] S4. Electrophoresis detection:

[0066] Prepare an agarose gel with a concentration of 1.5%. After the gel solidifies, pipette 1 μL of the PCR product, mix it with 1 μL of Loading buffer, load the sample, place it in an electrophoresis instrument, and run it at 110 V and 400 mA for 40 minutes. Observe the bright band situation in the gel imager to preliminarily judge the presence of the target species tilapia. As Figure 5 shown, using 4 pairs of primers to amplify the natural water sample of tilapia, 3 bands can be seen. The amplification efficiency of the original natural water sample is better than that of the 10-fold diluted natural water. Since the bp numbers of the primers SEQ ID NO.1-2 and SEQ ID NO.7-8 are similar, they cannot be distinguished by the gel image. It can be seen that the amplification efficiency of tissue samples > the amplification efficiency of aquaculture water > the amplification efficiency of natural water.

[0067] S5. Sequencing verification: Recover and purify the multiplex PCR amplification products using a DNA purification and recovery kit, send the purified sample to a biotechnology company for sequencing, and perform BLAST alignment analysis on the obtained sequences.

Claims

1. A primer set for detecting invasive species tilapia in water based on environmental DNA, characterized in that: The primer set includes 4 sets, and the sequences of the 4 primer sets are shown as SEQ ID NO.1-8.

2. The primer set according to claim 1, characterized in that: The 4 sets of primers and their corresponding regions are: The sequence of the first primer set is shown in SEQ ID NO. 1-2, which is used to amplify the 16Sribosomal RNA interval in tilapia mitochondria; The sequences of the second primer set are shown in SEQ ID NO. 3-4, and are used to amplify the ND1-tRNA-Ile interval in tilapia mitochondria; The sequences of the third primer set are shown in SEQ ID NO. 5-6, and are used to amplify the COI interval in tilapia mitochondria; The sequences of the fourth primer set are shown in SEQ ID NO. 7-8, and are used to amplify the D-loop region in tilapia mitochondria.

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

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

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

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

7. According to claim 1, it is characterized in that: All four primer sets can detect Nile tilapia, Tilapia zebrini and Oreochromis aureus.

8. 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.

9. 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 tilapia; (2) Prepare products for identifying or assisting in the identification of tilapia.

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