A method for detecting the presence of environmental DNA in catfish species

Abstract

The invention discloses a method for detecting environmental DNA of the presence of catfish species, and relates to the field of biological detection technology. The method for detecting environmental DNA of the presence of catfish species of the invention comprises the following steps: filtering and enriching a water environment sample; extracting DNA of the water environment sample to be tested; using catfish-specific primers to perform PCR amplification on the DNA of the water environment sample; judging whether catfish are present in the environmental sample to be tested based on whether specific amplification occurs in the electrophoresis result; the catfish-specific primer sequence is as follows: upstream primer 12F: 5'-CAAAGGTTTGGTCCTGACTTTA-3', downstream primer 12R: 5'-TGTCTCAGAGCCTGGTTCAAAA-3'. If a band of 800-850bp appears in the electrophoresis result, it indicates that catfish are present in the water environment sample to be tested.

Description

Technical Field

[0001] The invention belongs to the field of biological detection technology, and more specifically, relates to a method for detecting environmental DNA of catfish species. Background Art

[0002] The genus Corydoras belongs to the family Callichthyidae in the order Siluriformes. Currently, the genus Corydoras contains 175 valid species, making it the genus with the most species in the family Callichthyidae. Corydoras fish were originally distributed in South America and lived in rivers. Corydoras fish have existed on the earth for a long time, and they are small in size and swim slowly, but they can still survive today because they have strong vitality. Corydoras fish are benthic fish, they have strong disease resistance, strong appetite, are not picky about bait but have strong ability to compete for food, and only become active when eating, and usually hide in hiding places to rest.

[0003] There are a large number of catfish in the ornamental fish market in South my country. They are small in size, strong in vitality, strong in disease resistance, can remove algae, and are easy to raise. They are loved by ornamental fish enthusiasts everywhere. Currently, catfish have appeared in river fish surveys and wild fish fishing in South China. Catfish in the wild waters may escape and be released into wild waters.

[0004] Environmental DNA technology refers to a biological monitoring method that directly extracts total DNA from environmental samples, uses specific or universal primers for polymerase chain reaction (PCR), and uses high-throughput sequencing and bioinformatics analysis to identify target species sequences in the environment. This technology was first applied to soil microbiology research. Compared with traditional survey methods, environmental DNA technology has the advantages of being non-destructive, sensitive and efficient, and covering a large area. It does not require special equipment or a long period of observation time to identify small and small species in the environment. Environmental DNA technology has been widely used in research on biodiversity, endangered species, and alien invasive species. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for detecting environmental DNA of the presence of catfish species, which is used to detect whether catfish of the genus exist in the water environment.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for detecting environmental DNA for the presence of a species of the genus Catfish, comprising the steps of:

[0008] 1) Filter and enrich water environment samples;

[0009] 2) Extracting DNA from water environment samples to be tested;

[0010] 3) PCR amplification of DNA from water samples using primers specific to catfish;

[0011] 4) judging whether there is a fish of the genus Catfish in the environmental sample to be tested based on whether specific amplification occurs in the electrophoresis result;

[0012] The specific primer sequence of the catfish genus is as follows:

[0013] Upstream primer 12F: 5'-CAAAGGTTTGGTCCTGACTTTA-3',

[0014] Downstream primer 12R: 5′-TGTCTCAGAGCCTGGTTCAAAA-3′.

[0015] If a band of 800-850 bp appears in the electrophoresis result, it indicates that there are fish of the genus Catfish in the water environment sample to be tested; if no specific amplification occurs in the electrophoresis result, there are no fish of the genus Catfish in the water environment sample to be tested.

[0016] The PCR reaction system is: 5 μL 2×Taq Plus Master Mix II, 2 μL sterile water, 1 μL template DNA, 1 μL upstream primer, and 1 μL downstream primer.

[0017] The PCR reaction conditions are as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 36 cycles; and finally extension at 72°C for 10 min and storage at 10°C.

[0018] Specific primers for detecting the presence of catfish species in environmental samples, the specific primer sequences are as follows:

[0019] Upstream primer 12F: 5'-CAAAGGTTTGGTCCTGACTTTA-3',

[0020] Downstream primer 12R: 5′-TGTCTCAGAGCCTGGTTCAAAA-3′.

[0021] A test reagent or test kit containing specific primers for detecting the presence of a species of the genus Catfish in an environmental sample.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1) The method for detecting environmental DNA of the presence of catfish species of the present invention comprises the following steps: filtering and enriching a water environment sample; extracting DNA of the water environment sample to be tested; using catfish-specific primers to perform PCR amplification on the DNA of the water environment sample; and judging whether catfish of the genus exist in the environmental sample to be tested based on whether specific amplification occurs in the electrophoresis result.

[0024] 2) The specific primers for the genus Catfish provided by the present invention have high sensitivity and good stability, and the target fragment DNA sequence has species specificity and can identify the genus Catfish. The primer sequence is as follows:

[0025] Upstream primer 12F: 5'-CAAAGGTTTGGTCCTGACTTTA-3',

[0026] Downstream primer 12R: 5′-TGTCTCAGAGCCTGGTTCAAAA-3′.

[0027] 3) The size of the fragment amplified by the specific primers for the genus Catfish provided by the present invention is 800-850bp. If a band of 800-850bp appears in the electrophoresis result, it indicates that the genus Catfish exists in the water environment sample to be tested; if the electrophoresis result does not produce specific amplification, the genus Catfish does not exist in the water environment sample to be tested. The results show that the specific primers for the genus Catfish can detect the residual DNA of the genus Catfish in the water environment sample.

[0028] 4) The kit for detecting the presence of catfish species in environmental samples provided by the present invention can detect catfish species in water environment samples, and can quickly and accurately identify the presence of catfish in the water environment, providing an effective technical means for monitoring the invasion of catfish. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The electrophoresis diagram of the amplification products of the PCR primer specificity test of the genus Catfish (M is 2000bp DNA ladderMarker; Lane 1 is Corydoras paleatus; Lane 2 is Corydoras julii; Lane 3 is Corydoras aeneus; Lane 4 is Corydoras panda; Lane 5 is Rhinogobius zhoui; Lane 6 is Sicyopus zosterophorus; Lane 7 is Stiphodon semoni; Lane 8 is Pseudacanthicus leopardus; Lane 9 is Pethia conchonius; Lane 10 is the negative control (pure water));

[0030] Figure 2 This is the electrophoresis diagram of the amplification products of water samples using PCR primers for the genus Catfish (M is 2000bp DNA ladder marker; lane 1 is the positive control (Corydoras paleatus); lanes 2-9 are Corydoras paleatus, Corydoras aeneus, Corydoras trilineatus, Corydoras julii, Corydoras panda, Monodactylus sebae, Monodactylus argenteus, Hippichthys heptagonus; lane 10 is the negative control (pure water)). DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0032] The test fishes used in this application, including Sichuan pepper goby, leopard goby, coffee goby, panda goby, Zhou's goby, ring-banded cucumber goby, blue-faced electric goby, emperor blood diamond, rose barb, three-line leopard goby, bat pomfret, yellowfin pomfret, and red-bellied sea dragon, were all purchased from New World of Flowers, Birds, Fishes and Insects in Fangcun, Guangzhou City, Guangdong Province.

[0033] Morphological identification was performed according to the description obtained from FishBase (https: / / www.fishbase.se / search.php), and a small amount of caudal fin was clipped for identification using DNA barcoding.

[0034] Example 1

[0035] 1. Primer design and synthesis

[0036] Based on the 12s rRNA gene sequence of catfish, we searched for conserved sequence regions, designed upstream and downstream primers at both ends of the appropriate fragment, and after testing their specificity, they were synthesized by Nanjing Qingke Biotechnology Co., Ltd. The primer sequences are as follows:

[0037] Upstream primer 12F: 5'-CAAAGGTTTGGTCCTGACTTTA-3' (SEQ ID NO. 1),

[0038] Downstream primer 12R: 5'-TGTCTCAGAGCCTGGTTCAAAA-3' (SEQ ID NO. 2).

[0039] 2. Sensitivity and specificity of primers for catfish

[0040] The TaKaRa universal DNA extraction kit (Takara Biotech, Code No. 9765) was used to extract DNA from the test fish species, including Zanthoxylum bungeana, Leopard bungeana, Coffee bungeana, Panda bungeana, Zhou's goby, Annular cucumber goby, Blue-faced electric goby, Emperor blood diamond, and Rose barb. DNA was extracted according to the instructions.

[0041] The PCR reaction system (10 μL) was: 5 μL 2×Taq Plus Master MixⅡ (Dye Plus), 2 μL sterile water, 1 μL template DNA, 1 μL upstream primer, and 1 μL downstream primer.

[0042] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 36 cycles; final extension at 72°C for 10 min, and storage at 10°C.

[0043] The PCR products were electrophoresed on 1.0% agarose gel, and the electrophoresis results were photographed and stored in a gel imaging system.

[0044] 3. Sensitivity and specificity of primers for catfish

[0045] The results are as follows Figure 1 As shown: M is a 2000bp DNA ladder marker; Lane 1 is Corydoraspaleatus; Lane 2 is Corydoras julii; Lane 3 is Corydoras aeneus; Lane 4 is Corydoras panda; Lane 5 is Rhinogobius zhoui; Lane 6 is Sicyopus zosterophorus; Lane 7 is Stiphodon semoni; Lane 8 is Pseudacanthicus leopardus; Lane 9 is Pethia conchonius; Lane 10 is a negative control (pure water). Only the Corydoras julii, Corydoras julii, Corydoras aeneus and Corydoras panda of the genus Corydoras amplified the 800-850bp band, and no other samples showed the 800-850bp band.

[0046] The results showed that this primer had strong specificity.

[0047] Example 2

[0048] 1. Filtration and enrichment of water environment samples

[0049] Eight species of fish for testing, including Chinese prickly ash, coffee prickly ash, three-line leopard prickly ash, leopard prickly ash, panda prickly ash, bat pomfret, yellowfin pomfret, and red-bellied sea dragon, were kept in 2L simulated ecological fish tanks. After the fish adapted to the breeding environment, they were cultured without changing the water for 10 days. 500mL water samples were collected from the upper, middle, and lower layers of each fish tank and stored in an incubator. The upper, middle, and lower water samples in each fish tank were mixed as one sample, and the water sample where Chinese prickly ash lived was used as a positive control. Then the other 8 groups of samples were tested, and a mixed cellulose filter membrane with a pore size of 0.22μm was used to complete the filtration within 6h. The filter membrane was placed in a 1.5mL sterile cryopreservation tube and frozen at -20℃. A negative control (pure water) was set during the filtration to identify whether there was exogenous DNA contamination. All experimental equipment was soaked and disinfected with 10% bleach solution for 30min before and after use to remove residual DNA, and then fully rinsed with sterile water.

[0050] 2. DNA extraction of water environment samples to be tested

[0051] The filter membrane was cut into pieces with sterile scissors, and environmental DNA was extracted using the TaKaRa universal DNA extraction kit (Takara Biotech, Code No. 9765) according to the instructions.

[0052] 3. Use the specific primers of catfish to detect water environment samples

[0053] The primer sequences are as follows:

[0054] Upstream primer 12F: 5'-CAAAGGTTTGGTCCTGACTTTA-3',

[0055] Downstream primer 12R: 5′-TGTCTCAGAGCCTGGTTCAAAA-3′.

[0056] The size of the amplified fragment is 800-850bp. If a band of 800-850bp appears in the electrophoresis result, it indicates that there are fish of the genus Catfish in the water environment sample to be tested; if no specific amplification occurs in the electrophoresis result, there are no fish of the genus Catfish in the water environment sample to be tested.

[0057] The PCR reaction system (10 μL) was: 5 μL 2×Taq Plus Master MixⅡ (Dye Plus), 2 μL sterile water, 1 μL template DNA, 1 μL upstream primer, and 1 μL downstream primer.

[0058] The PCR reaction conditions were as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 36 cycles; and finally 72°C extension for 10 min, and storage at 10°C. The PCR product was electrophoresed on a 1.0% agarose gel, and the electrophoresis results were photographed and stored in a gel imaging system. The presence of the genus Catfish in the water environment was determined based on the presence or absence of the electrophoresis bands.

[0059] 4. Detection effect of primers on water environment samples

[0060] The results are as follows Figure 2 As shown in the figure, M is a 2000bp DNA ladder marker; lane 1 is a positive control (DNA of Chinese prickly ash); lanes 2-9 are water samples of Chinese prickly ash Corydoras paleatus, coffee ash Corydoras aeneus, three-line leopard ash Corydoras trilineatus, leopard ash Corydoras julii, panda ash Corydoras panda, bat pomfret Monodactylus sebae, yellowfin pomfret Monodactylus argenteus, and red-bellied sea dragon Hippichthysheptagonus; lane 10 is a negative control (pure water). Lanes 1-6 all have bands of 800-850bp, and lanes 7-10 have no bands, so the experimental results are reliable.

[0061] The results show that the Armored Catfish-specific primers provided by the present invention can detect the residual DNA of Armored Catfish in water environment samples.

[0062] Example 3

[0063] A kit for screening fish of the genus Catfish in water environment samples, the kit comprising primer sequences as shown below:

[0064] Upstream primer 12F: 5'-CAAAGGTTTGGTCCTGACTTTA-3',

[0065] Downstream primer 12R: 5′-TGTCTCAGAGCCTGGTTCAAAA-3′.

[0066] The results show that the kit for screening catfishes in water environment samples provided by the present invention can detect catfishes in water environment samples.

[0067] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A method for detecting the presence of environmental DNA of a species of the genus Catfish, characterized in that the steps include: 1) Filter and enrich water environment samples; 2) Extracting DNA from water environment samples to be tested; 3) PCR amplification of DNA from water samples using primers specific to catfish; 4) judging whether there is a fish of the genus Catfish in the environmental sample to be tested based on whether specific amplification occurs in the electrophoresis result; The specific primer sequence of the catfish genus is as follows: Upstream primer 12F: 5'-CAAAGGTTTGGTCCTGACTTTA-3', Downstream primer 12R: 5′-TGTCTCAGAGCCTGGTTCAAAA-3′.

2. The method for detecting the presence of environmental DNA of a species of the genus Catfish according to claim 1, characterized in that: If a band of 800-850 bp appears in the electrophoresis result, it indicates that there are fish of the genus Catfish in the water environment sample to be tested; if no specific amplification occurs in the electrophoresis result, there are no fish of the genus Catfish in the water environment sample to be tested.

3. The method for detecting the presence of environmental DNA of a species of the genus Catfish according to claim 1, characterized in that: The PCR reaction system is: 5 μL 2×Taq Plus Master Mix II, 2 μL sterile water, 1 μL template DNA, 1 μL upstream primer, and 1 μL downstream primer.

4. The method for detecting the presence of environmental DNA of a species of the genus Catfish according to claim 1, characterized in that: The PCR reaction conditions are as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 36 cycles; and finally extension at 72°C for 10 min and storage at 10°C.

5. A specific primer for detecting the presence of a species of the genus Catfish in an environmental sample, characterized in that: The specific primer sequences are as follows: Upstream primer 12F: 5'-CAAAGGTTTGGTCCTGACTTTA-3', Downstream primer 12R: 5′-TGTCTCAGAGCCTGGTTCAAAA-3′.

6. A detection reagent or a detection kit containing the specific primers for detecting the presence of a species of the genus Catfish in an environmental sample as claimed in claim 5.

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