Primer group, kit and detection method for detecting freshwater fish in water environment

Abstract

The invention relates to the technical field of biology, in particular to a primer group, a kit and a detection method for detecting freshwater fish in a water environment. The invention provides a primer group for detecting freshwater fish in a water environment. The sequence of the primer group is as shown in SEQ ID NO: 1-SEQ ID NO: 2. The macro barcode primer group provided by the invention can improve the accuracy of freshwater fish resource monitoring, effectively improve the accuracy of freshwater fish environment diversity detection, and meet the demand of accurate monitoring of freshwater fish resources in actual ecological detection work.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a primer set, a kit and a detection method for detecting freshwater fish in a water environment. Background Art

[0002] Ecological monitoring, as an important means of measuring the interrelationships between life systems and evaluating the level of species diversity, plays a key role in the protection of rare aquatic organisms, the prevention and control of invasive species, the assessment of ecological health, and the evaluation of fish population structure. However, traditional ecological monitoring methods such as electronic tag implantation and catch statistics are facing problems such as high cost, low accuracy, and great destructiveness, and have gradually been unable to meet the current needs of protection work for accurately monitoring the ecological status of river basins and implementing effective biodiversity assessments.

[0003] At the same time, with the development of monitoring technologies, molecular-level ecological monitoring technologies represented by environmental nucleic acid (eNA; including environmental DNA and environmental RNA) technologies have become important methods for evaluating fish diversity and ecological risks in river basins due to their non-invasive and high-accuracy characteristics, and play an important guiding role in the formulation and implementation of relevant protection work. Currently, according to different amplification regions, a variety of environmental DNA (eDNA) barcode technologies have been disclosed (CN109825563A, CN104531879A, CN115948500A, etc.), verifying the application potential of related technologies. As an important technical supplement, environmental RNA (eRNA) technology has been developed and disclosed in recent years: CN114981449A discloses a method for investigating a water environment ecosystem using eRNA, effectively reducing false positive results caused by the difficulty of DNA degradation. However, since this work mainly uses Mifish-U and Mifish-E for library construction, these primers are mainly designed based on seawater fish information and are not optimized for eRNA detection, resulting in low accuracy in freshwater fish eRNA detection. Considering that the work of protecting the Yangtze River mainly focuses on freshwater fish, the current primer design cannot meet the needs of accurately monitoring the resources of Yangtze River freshwater fish in current actual ecological detection work. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defect of low detection accuracy of freshwater fish in the water environment in the prior art, so as to provide a primer set, a kit and a detection method for detecting freshwater fish in the water environment.

[0005] To this end, in a first aspect, the present invention provides a primer set for detecting freshwater fish in a water environment, and the sequences of the primer set are shown as SEQ ID NO: 1-SEQ ID NO: 2.

[0006] In some embodiments, the primer set for detecting freshwater fish in a water environment is designed based on a conserved region of freshwater fish, and the sequence of the conserved region of freshwater fish is shown as SEQ ID NO: 16-SEQ ID NO: 17.

[0007] In a second aspect, the present invention provides a kit for detecting freshwater fish in a water environment, including the above-mentioned primer set for detecting freshwater fish in a water environment.

[0008] In some embodiments, the kit further includes DNA polymerase, deoxynucleoside triphosphate, buffer, and nuclease-free water.

[0009] Preferably, the buffer includes a reaction buffer and a high CG buffer.

[0010] In a third aspect, the present invention provides a method for detecting freshwater fish in a water environment, including the following steps: extracting environmental nucleic acids from a sample to be detected; performing PCR amplification on the extracted environmental nucleic acids using the above-mentioned primer set for detecting freshwater fish in a water environment or the above-mentioned kit for detecting freshwater fish in a water environment; obtaining a detection result by performing high-throughput sequencing on the PCR amplification product.

[0011] In some embodiments, the environmental nucleic acids include at least one of eRNA and eDNA.

[0012] In some embodiments, the reaction program of the PCR amplification is: after 3 minutes at 95°C, perform 35 cycles consisting of 30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C, and then extend for 5 minutes at 72°C.

[0013] In some embodiments, for the reaction system of the PCR amplification, with a volume of 25 μL, 0.25 μL of DNA polymerase, 5 μL of 5× reaction buffer, 5 μL of 5× high GC buffer, 2 μL of dNTP (10 mM), 2 μL of cDNA, 1 μL each of forward and reverse primers (10 μM), and 8.75 μL of nuclease-free water, wherein the concentration of dNTP is 10 mM, and the concentrations of the forward primer and the reverse primer are 10 μM.

[0014] Preferably, the PCR reaction system (25 μL) is 0.25 μL of Q5 high-fidelity DNA polymerase, 5 μL of 5×Reaction Buffer, 5 μL of 5×High GC Buffer, 2 μL of dNTP (10 mM), 2 μL of cDNA, 1 μL each of forward / reverse primers (10 μM), and 8.75 μL of nuclease-free water.

[0015] In some of these embodiments, before extracting the environmental nucleic acids of the sample to be detected, it further includes the step of pre-treating the sample to be detected. The specific steps include collecting the water sample and filtering, and the filtering uses a 45-μm mixed cellulose ester membrane.

[0016] Meanwhile, the present invention provides the application of the above-mentioned primer set for detecting freshwater fish in the water environment, or the above-mentioned kit for detecting freshwater fish in the water environment, or the above-mentioned detection method for freshwater fish in the water environment in freshwater fish resource monitoring.

[0017] The technical solution of the present invention has the following advantages:

[0018] A primer set for detecting freshwater fish in the water environment provided by the present invention, the sequences of the primer set are shown as SEQ ID NO: 1-SEQ ID NO: 2. The metabarcoding primer set provided by the present invention can improve the accuracy of freshwater fish resource monitoring, effectively improve the accuracy of detecting the environmental diversity of freshwater fish, and meet the requirements for accurate monitoring of freshwater fish resources in actual ecological detection work. Description of the Drawings

[0019] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0020] Figure 1 It is the gel electrophoresis pattern of the PCR amplification reaction product based on eRNA with the primer name 12r278 in Example 2 of the present invention;

[0021] Figure 2 It is the gel electrophoresis pattern of the PCR amplification reaction product based on eRNA with the primer name 12r638 in Example 2 of the present invention;

[0022] Figure 3 It is the gel electrophoresis pattern of the PCR amplification reaction product based on eRNA with the primer name 12r814-1 in Example 2 of the present invention;

[0023] Figure 4 It is the gel electrophoresis map of the PCR amplification reaction product based on eRNA with the primer name 12r814-2 in Example 2 of the present invention;

[0024] Figure 5 It is the gel electrophoresis map of the PCR amplification reaction product based on eRNA with the primer name 12r1007 in Example 2 of the present invention;

[0025] Figure 6 It is the gel electrophoresis map of the PCR amplification reaction product based on eRNA with the primer name 16r1158 in Example 2 of the present invention;

[0026] Figure 7 It is the gel electrophoresis map of the PCR amplification reaction product based on eRNA with the primer name 16r1279 in Example 2 of the present invention;

[0027] Figure 8 It is the gel electrophoresis map of the PCR amplification reaction product based on eRNA with the primer name MiFish-E in Example 2 of the present invention;

[0028] Figure 9 It is the gel electrophoresis map of the PCR amplification reaction product based on eRNA with the primer name MiFish-U in Example 2 of the present invention;

[0029] Figure 10 It is the gel electrophoresis map of the PCR amplification reaction product based on eDNA with the primer name 12r278 in Example 2 of the present invention;

[0030] Figure 11 It is the gel electrophoresis map of the PCR amplification reaction product based on eDNA with the primer name 12r638 in Example 2 of the present invention;

[0031] Figure 12 It is the gel electrophoresis map of the PCR amplification reaction product based on eDNA with the primer name 12r814-1 in Example 2 of the present invention;

[0032] Figure 13 It is the gel electrophoresis map of the PCR amplification reaction product based on eDNA with the primer name 12r814-2 in Example 2 of the present invention;

[0033] Figure 14 It is the gel electrophoresis map of the PCR amplification reaction product based on eDNA with the primer name 12r1007 in Example 2 of the present invention;

[0034] Figure 15 It is the gel electrophoresis map of the PCR amplification reaction product based on eDNA with the primer name 16r1158 in Example 2 of the present invention;

[0035] Figure 16 It is the gel electrophoresis diagram of the PCR amplification reaction product based on eDNA with the primer name 16r1279 in Example 2 of the present invention;

[0036] Figure 17 It is the gel electrophoresis diagram of the PCR amplification reaction product based on eDNA with the primer name MiFish-E in Example 2 of the present invention;

[0037] Figure 18 It is the gel electrophoresis diagram of the PCR amplification reaction product based on eDNA with the primer name MiFish-U in Example 2 of the present invention;

[0038] Figure 19 It is the accuracy rate of each primer based on eRNA detection in Example 2 of the present invention;

[0039] Figure 20 It is the accuracy rate of each primer based on eDNA detection in Example 2 of the present invention. Detailed implementation manners

[0040] The following embodiments are provided to better further understand the present invention. It is not limited to the described best implementation manner, and does not limit the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other existing technologies falls within the protection scope of the present invention.

[0041] For those not specifying specific experimental steps or conditions in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For those reagents or instruments not specifying the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0042] Example 1

[0043] This example provides a design method for a primer set for detecting freshwater fish in a water environment. The specific steps and parameters are as follows:

[0044] Sort out the mitochondrial genome information of all fish in the MitoFish public database, and screen the information of freshwater fish and brackish water fish based on the NCBI species classification database. A total of 1707 species of freshwater / brackish water fish are obtained.

[0045] Subsequently, the Clustal Omega tool was used to align the rRNA coding regions (12s and 16s rRNA regions) of the target fish, and the conserved regions and variable regions were screened. The specific information of the conserved regions is shown in Table 1. Considering their conservation, discrimination, and amplification length, a total of 7 pairs of primers were designed for each region of 12s rRNA and 16s rRNA. The sequence names and corresponding information are shown in Table 2.

[0046] Table 1 Information of conserved regions

[0047]

[0048]

[0049] It should be noted that the forward primer of primer 12R278 was designed according to the conserved region sequence of SEQ ID NO:16, the reverse primer of primer 12R278 was designed according to the conserved region sequence of SEQ ID NO:17, the forward primer of primer 12R638 was designed according to the conserved region sequence of SEQ ID NO:18, the reverse primer of primer 12R638 and the reverse primer of 12R814-1 were designed according to the conserved region sequence of SEQ ID NO:19, the forward primers of primer 12R814-1 and 12R814-2 were designed according to the conserved region sequence of SEQ ID NO:20, the forward primer of primer 12R1007 was designed according to the conserved region sequence of SEQ ID NO:21, the reverse primers of primer 12R814-2 and 12R1007 were designed according to the conserved region sequence of SEQ ID NO:22, the forward primer of primer 16R1158 was designed according to the conserved region sequence of SEQ ID NO:23, the reverse primer of primer 16R1158 and the forward primer of 16R1279 were designed according to the conserved region sequence of SEQ ID NO:24, and the reverse primer of primer 16R1279 was designed according to the conserved region sequence of SEQ ID NO:25.

[0050] Table 2 Primer information

[0051]

[0052] Example 2

[0053] This example provides a method for detecting freshwater fish in the water environment. The specific steps and parameters are as follows:

[0054] (1) Sampling: Common freshwater / brackish water fish in the upper and lower reaches of the Yangtze River were used as monitoring objects, and the information of the monitoring objects is shown in Table 3. Each monitoring object in Table 2 was cultured in an aquarium. When sampling, the circulating water system in each aquarium was turned off, and a water sampler was used to obtain 500 mL of water samples at the central position of each aquarium. Then the water samples in each aquarium were mixed to obtain a mixed solution. The mixed solution was filtered through a 45 μm mixed fiber filter membrane to obtain 5 L of water samples, and the filter membrane was stored in an ep tube. The ep tube was frozen in an environment of -80 °C.

[0055] Table 3 Information of Monitoring Objects

[0056]

[0057]

[0058] (2) eRNA Extraction / Detection Method:

[0059] Extract the nucleic acid fragments enriched on the filter membrane stored in the ep tube. Use the RNeasy PowerSoil Total RNA Kit to recover eRNA to obtain eRNA samples. Subsequently, for the eRNA samples, use III 1stStrand cDNA Synthesis Kit(+gDNAwiper) kit to synthesize cDNA templates.

[0060] Send the template cDNA to Personal Biotechnology Co., Ltd. for sequencing (sequencing machine: illumina novaseq 6000, sequencing mode: PE250). Synthesize the relevant primers for primer numbers 1 - 7 in Table 2 respectively, and perform PCR amplification on the sample cDNA to produce an amplicon library and use gel electrophoresis to determine the amplification effect. Among them, the PCR reaction procedure is as follows: after 3 minutes at 95 °C, perform 35 cycles consisting of 30 seconds at 95 °C, 30 seconds at 60 °C, and 30 seconds at 72 °C, and then finally extend for 5 minutes at 72 °C;

[0061] The PCR reaction system (25 μL) is 0.25 μL of Q5 high-fidelity DNA polymerase, 5 μL of 5×Reaction Buffer, 5 μL of 5×High GC Buffer, 2 μL of dNTP (10 mM), 2 μL of cDNA, 1 μL each of forward / reverse primers (10 μM), and 8.75 μL of nuclease-free water.

[0062] The results are shown in Figures 1 - 7 , Figures 1 - 7 where M represents the marker, 1 represents the amplification products of each primer pair, and CK represents deionized water.

[0063] (3) eDNA extraction / detection method:

[0064] Extract the nucleic acid fragments enriched on the filter paper stored in the EP tube, and use the CTAB method to recover eDNA to obtain the eDNA template.

[0065] Send the template eDNA to Personal Biotechnology Co., Ltd. for sequencing (sequencing machine: Illumina NovaSeq 6000, sequencing mode: PE250). Synthesize the relevant primers with the primer numbers 1-7 in Table 2 respectively, and use the synthesized primers to perform PCR amplification on the sample eDNA template respectively, prepare the amplicon library and use gel electrophoresis to determine the amplification effect. Among them, the PCR reaction program is: after 3 minutes at 95°C, perform 35 cycles consisting of 30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C, and then finally extend for 5 minutes at 72°C;

[0066] The PCR reaction system (25 μL) is: 0.25 μL of Q5 high-fidelity DNA polymerase, 5 μL of 5×Reaction Buffer, 5 μL of 5×High GC Buffer, 2 μL of dNTP (10 mM), 2 μL of DNA, 1 μL each of forward / reverse primers (10 μM), and 8.75 μL of nuclease-free water.

[0067] The results are shown in Figures 10 - 18 , Figures 10 - 18 where M represents the marker, 1 represents the amplification products of each primer pair, and CK represents deionized water.

[0068] (4) According to the methods in steps (2) and (3) respectively, using the two pairs of primers provided by the patent publication number CN114981449A, namely MiFish-E-F and MiFish-E-R, MiFish-U-F and MiFish-U-R as the control groups, perform PCR amplification on the sample cDNA template and eDNA template respectively, prepare the amplicon library and use gel electrophoresis to determine the amplification effect. The specific information of the primers is shown in Table 2;

[0069] The PCR reaction program is: after 3 minutes at 95°C, perform 35 cycles consisting of 30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C, and then finally extend for 5 minutes at 72°C.

[0070] The PCR reaction system (25 μL) consists of 0.25 μL of Q5 high-fidelity DNA polymerase, 5 μL of 5× Reaction Buffer, 5 μL of 5× High GC Buffer, 2 μL of dNTP (10 mM), 2 μL of DNA, 1 μL each of forward / reverse primers (10 μM), and 8.75 μL of nuclease-free water.

[0071] The results are shown in Figure 8 、 Figure 9 、 Figure 19 and Figure 20 , Figure 8 、 Figure 9 、 Figure 19 and Figure 20 where M represents the marker, 1 represents the amplification products of each primer pair, and CK represents deionized water; among them, according to Figures 1 - 9 it can be seen that primer numbers 1-4 and 6-9 can all be amplified successfully, and the amplification products are obtained as the amplicon library, while the product length amplified by primer number 5 is too short to obtain an amplification result.

[0072] (5) Sequencing: The amplicon library obtained through steps (2)-(4) is provided to Personal Biotechnology Co., Ltd. for paired-end sequencing using the illumina novaseq 6000 platform and species annotation based on the NCBI freshwater fish database to obtain the species distribution in the water sample. Among them, the calculation method of accuracy is as follows: Accuracy (%) = {(The number of biological species belonging to the true positive group detected by eDNA or eRNA metabarcoding) / (The total number of biological species belonging to the true positive group)} × 100. In this example, the total number of biological species belonging to the true positive group is 39, and the results are shown in Table 4 and Table 5 and Figures 18 - 19 。

[0073] Considering that the water samples in the exhibition hall were collected from the Yangtze River (natural water body) and there are trace residues of other fish eNA, the fish annotation results with an absolute abundance less than 300 after sequencing are regarded as an inevitable error range and are not included in the calculation of later accuracy and positive quantity.

[0074] Table 4 Accuracy of each primer eRNA

[0075]

[0076]

[0077] Table 5 Accuracy of each primer eDNA

[0078]

[0079] There are 2-3 base mismatches between the two primer pairs MiFish-U and MiFish-E provided by the prior art and the conserved region of the 12s rRNA of freshwater fish (the mismatched bases are shown as bold bases in Table 2). There is a possibility of non-specific amplification during the actual eRNA monitoring library construction process, which may be the reason for the low accuracy of freshwater fish eRNA detection.

[0080] As can be seen from Table 4, 27 and 22 true positive species were respectively detected using primer numbers 8 and 9, and the accuracy rates were 69.2% and 56.4% respectively, which were basically consistent with the publicly disclosed data in CN114981449A. Since the 12r278 primer was designed and optimized based on the conserved region sequence of freshwater fish, the mismatched bases were corrected and the lengths of the forward and reverse primers were increased, improving the accuracy of amplicon library construction. Finally, 34 true positive species were successfully identified, and the accuracy rate reached 87.2%, showing a significant improvement compared with the existing primers. For the primers 12r638, 12r814-1, 12r814-2, 16r1158, and 16r1279 targeting other regions of rRNA, 19, 17, 10, 18, and 19 true positive species were respectively identified, and the accuracy rates were 48.7%, 43.6%, 25.6%, 46.2%, and 48.7% respectively. The accuracy rates decreased slightly compared with the prior art, which may be due to the insufficient conservation of other regions.

[0081] As can be seen from Table 5, in eDNA detection, all primers can be used to construct libraries and perform species annotation, demonstrating the usefulness of the primers. At the same time, the primer 12r278 still maintains a high accuracy rate in eDNA detection. Finally, 31 true positive species were successfully identified, and the accuracy rate reached 71.8%, which is at the same level as MiFish-E and MiFish-U. For the primers 12r638, 12r814-1, 12r814-2, 16r1158, and 16r1279 targeting other regions of rRNA, 17, 16, 18, 16, and 21 true positive species were respectively identified, and the accuracy rates were 43.6%, 41.0%, 46.2%, 53.8%, respectively. Although the accuracy rates decreased slightly compared with the 12r278 primer, the different characteristics of these primers in the targeted regions can provide more choices in the monitoring work, ensuring the applicability and redundancy of the technology. Finally, through the application in eDNA monitoring, the usability of the primers designed in the present invention in eDNA fish diversity monitoring was verified.

[0082] Obviously, the above embodiments are merely examples given for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A primer set for detecting freshwater fish in a water environment, characterized in that, The sequences of the primer sets are shown as SEQ ID NO: 1 - SEQ ID NO:

2.

2. The primer set for detecting freshwater fish in a water environment according to claim 1, characterized in that, The primer set for detecting freshwater fish in the water environment is designed based on the conserved regions of freshwater fish, and the sequences of the conserved regions of freshwater fish are shown as SEQ ID NO: 16 - SEQ ID NO:

17.

3. A kit for detecting freshwater fish in a water environment, characterized in that, It includes the primer set for detecting freshwater fish in the water environment according to any one of claims 1 - 2.

4. The kit for detecting freshwater fish in a water environment according to claim 3, characterized in that, The kit further includes DNA polymerase, deoxynucleoside triphosphates, buffer, and nuclease - free water.

5. A detection method for freshwater fish in a water environment, characterized in that, It includes the following steps, Extract the environmental nucleic acids of the sample to be detected; Perform PCR amplification on the extracted environmental nucleic acids using the primer set for detecting freshwater fish in the water environment according to any one of claims 1 - 2 or the kit for detecting freshwater fish in the water environment according to any one of claims 3 - 4; Obtain the detection result by performing high - throughput sequencing on the PCR amplification product.

6. The detection method of freshwater fish in the water environment according to claim 5, characterized in that, The environmental nucleic acids include at least one of eRNA and eDNA.

7. The detection method of freshwater fish in the water environment according to claim 5, characterized in that, The reaction program of the PCR amplification is as follows: after 3 minutes at 95°C, perform 35 cycles consisting of 30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C, and then extend for 5 minutes at 72°C.

8. The detection method of freshwater fish in the water environment according to claim 5, characterized in that, For the reaction system of the PCR amplification, based on a volume of 25 μL, 0.25 μL of DNA polymerase, 5 μL of 5× reaction buffer, 5 μL of 5× high - GC buffer, 2 μL of dNTP, 2 μL of cDNA, 1 μL each of the forward and reverse primers, 8.75 μL of nuclease - free water, wherein, the concentration of dNTP is 10 mM, and the concentrations of the forward primer and the reverse primer are 10 μM.

9. The detection method of freshwater fish in the water environment according to claim 5, characterized in that, Before extracting the environmental nucleic acids of the sample to be detected, it further includes the step of pre - treating the sample to be detected. The specific steps include collecting the water sample and filtering, and the filtering uses a 45 - μm mixed - fiber filter membrane.

10. The application of the primer set for detecting freshwater fish in the water environment according to any one of claims 1 - 2, or the kit for detecting freshwater fish in the water environment according to any one of claims 3 - 4, or the detection method of freshwater fish in the water environment according to any one of claims 5 - 9 in freshwater fish resource monitoring.

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