A gene chip for detecting Sanmen mud crabs

Abstract

The object of the present invention is to provide a gene chip for detecting Sanmen mud crabs. The present invention collects mud crabs from the main production areas of mud crab farming in the provinces along the southeast coast of China, performs 16S rRNA sequencing on their intestinal microbial genomes, and through comparative analysis, screens out the specific microbial populations in each main production area, and comprehensively designs the characteristic DNA sequences of these populations. Finally, molecular markers for identifying mud crabs in each area are obtained and developed into a gene chip with characteristics such as fast speed, high efficiency, and sensitivity. The origin of Sanmen mud crabs is identified through the gene chip, and non-Sanmen mud crabs are distinguished, thus solving the problem of identifying Sanmen mud crabs.

Description

Technical Field

[0001] The present invention belongs to the technical field of crab origin identification, and particularly relates to a gene chip for detecting Scylla paramamosain from Sanmen. Background Art

[0002] Scylla paramamosain belongs to Arthropoda, Malacostraca, Decapoda, Portunidae, Scylla. It is a warm-water and euryhaline marine crab with characteristics such as fast growth, large size, and strong adaptability. Its meat is delicious and nutritious. It is a traditional precious seafood product and also the main marine crab species for artificial breeding in many countries. In China, it is mainly distributed in Guangdong, Fujian, Zhejiang, Guangxi, Hainan and other places.

[0003] Sanmen County in Zhejiang is located on the coast of Sanmen Bay. Its hundreds of kilometers of rich coastline has created unique conditions for the breeding of Scylla paramamosain. Over the years, the popularity and influence of Scylla paramamosain (Sanmen Scylla paramamosain) bred in Sanmen have been continuously enhanced. It has been successively named the "Hometown of Chinese Scylla paramamosain" and the "China Research Base for Sanmen Scylla paramamosain". In 2006, Sanmen Scylla paramamosain passed the protection of national geographical indication products and became the only geographical indication protected seafood product in the country at that time. It also won many honors such as the title of Chinese famous brand agricultural product.

[0004] Although Sanmen Scylla paramamosain was on the list of the most influential agricultural products brands in Zhejiang Agricultural Expo in 2015, the current "mixed" phenomenon in the market has seriously affected the brand reputation of Sanmen Scylla paramamosain. How to strengthen the brand effect of Sanmen Scylla paramamosain, improve the quality of Sanmen Scylla paramamosain, enhance the competitiveness of the industry, and avoid being maliciously counterfeited are unavoidable issues for the long-term development of the Sanmen Scylla paramamosain industrial economy.

[0005] The intestine is the most important digestive and absorption organ of Scylla paramamosain, in which there are a large number and complex structure of microorganisms. They are interdependent and restrictive with the host and form a unique intestinal microecosystem in the long-term evolution process, which is closely related to the health of the host. The intestinal flora of aquatic animals is positively correlated with the host and the nature of the water environment. Their co-selection enables a specific flora structure to colonize the intestinal mucosa layer. The latest research found that the environment of the aquaculture water body and the diversity distribution of the water body microbial flora can affect the diversity distribution of the intestinal microbial flora of aquatic animals. There are differences between the aquaculture water environments in different regions, and the diversity distribution of the microbial flora in the aquaculture water body can directly affect the diversity distribution of the intestinal microbial flora of Scylla paramamosain. Therefore, there are differences in the diversity distribution of the intestinal microorganisms of Scylla paramamosain cultured in different regions. Summary of the Invention

[0006] The object of the present invention is to provide a gene chip for detecting Sanmen mud crabs, which can be used to identify mud crabs from different origins, thereby making up for the deficiencies of the prior art.

[0007] The present invention first provides a set of molecular markers, and the molecular markers are specific nucleotide fragments of Scylla paramamosain cultured in the areas of Sanmen, Ruian, Yunxiao, Shantou, Zhuhai, Yangjiang, Beihai, and Wanning.

[0008] Among them, the specific nucleotide fragment of Scylla paramamosain cultured in the Sanmen area has the following sequence (SEQ ID NO:1):

[0009] TGGGGAATATTGCACAATGGGGGAAACCCTGATCCAGCAATTCTGTGTGCACGATGAAGGTCTTCGGATTGTAAAGTGCTTTTAGGTGGGAAGAAGAAAGTGACGGTACCACCAGAAGAAGCGACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGAGTCTAGGCGGCTTGTTAAGTCAGATGTGAAAATGCGGGGCTCAACTCCGTATTGCGTTTGAAACTGGCATGCTAGAGTACTGGAGAGGTGGGCGGAACTACAAGTGTAGAGGTGAAATTCGTAGATATTTGTAGGAATGCCGATAGTGAAGACAGCTCACTGGACAGATACTGACGCTAAAGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCT;

[0010] Among them, the specific nucleotide fragment of Scylla paramamosain cultured in the Ruian area has the following sequence (SEQ ID NO:2):

[0011] TGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAAGGATGAAGGTCTTCGGATTGTAAACTTCTATCAGCAGGGAAGAATTATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCGAGCGTTATCCGGAATTACTGGGTGTAAAGGGTGCGTAGGCGGTAATGCAAGTCAGATGTGAAAACTCTGGGCTCAACCTAGAGGCTGCATCTGAAACTGCGTAACTAGAGTGCAGGAGAGGAAAGTGGAATTCCGAGTGTAGCGGTGAAATGCGTAGAGATTCGGAGGAACACCAGTAGCGAAGGCGACTTTCTGGACTGTAACTGACGCTGAGGCACGAAAGCGTGGGGAGCGAACAGGATTAGATACCCT;

[0012] The specific nucleotide fragment of Scylla paramamosain cultured in Yunxiao area has the following sequence (SEQ ID NO: 3):

[0013] TGGAGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCT;

[0014] Among them, the specific nucleotide fragment of Scylla paramamosain cultured in Shantou area, and its sequence is as follows (SEQ ID NO:4):

[0015] TGGGGAATATTGCGCAATGGGGGAAACCCTGACGCAGCAACGCCGCGTGAGTGAAGAAGGCCTTAGGGTTGTAAAGCTCTGTCATATGGGAAGATAATGACGGTACCATAAGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCGAGCGTTGTCCGGATTTACTGGGCGTAAAGGATGCGTAGGCGGATATTTAAGTGGGATGTGAAATACCCGAGCTCAACTCGGGTGCTGCATTCCAAACTGGATATCTAGAGTGTCGGAGAGGAAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGAGATTAGGAAGAACACCAGTGGCGAAGGCGGCTTTCTGGACGATAACTGACGCTGAGGCATGAAAGCGTGGGGAGCAAACAGGATTAGATACCCT;

[0016] Among them, the specific nucleotide fragment of Scylla paramamosain cultured in Zhuhai area, and its sequence is as follows (SEQ ID NO:5):

[0017] TAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAATGCCGCGTGAGCGATGAAGGCCTTCGGGTCGTAAAGCTCTGTCCTCAAGGAAGATAATGACGGTACTTGAGGAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCTAGCGTTATCCGGAATTACTGGGCGTAAAGGGTGCGTAGGTGGTTTTTTAAGTCAGAAGTGAAAGGCTACGGCTCAACCGTAGTAAGCTTTTGAAACTAGAGAACTTGAGTGCAGGAGAGGAGAGTAGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAATACCAGTAGCGAAGGCGGCTCTCTGGACTGTAACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCT;

[0018] Among them, the specific nucleotide fragment of Scylla paramamosain cultured in Yangjiang area, and its sequence is as follows (SEQ ID NO:6):

[0019] TGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTGAGGTTAATAACCTTATTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTGGAGTCTTGTAGAGGGGGGTAGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCT;

[0020] Among them, the specific nucleotide fragment of Scylla paramamosain cultured in Beihai area, and its sequence is as follows (SEQ ID NO:7):

[0021] TGGGGAATATTGGACAATGGGCCACAAGCCTGATCCAGCAATTCTGTGTGCACGATGAAGGTTTTCGGATCGTAAAGTGCTTTCAGTTGGGAAGAAGAAAGTGACGGTACCAACAGAAGAAGCGACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCGCAAGCGTTATCCGGATGTATTGGGCGTAAAGCGCGTCTAGGCGGAAAAGAAAGTCTGATGTTAAAATGCGGGGCTCAACTCCGTATTGCGTTGGAAACTGCTTTTCTAGAGTACTGGAGAGGTGGGCGGAACTACAAGTGTAGAGGTGAAATTCGTAGATATTTGTAGGAATGCCGATGGAGAAGTCAGCTCACTGGACAGATACTGACGCTAAAGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCT;

[0022] The specific nucleotide fragment of Scylla paramamosain cultured in Wanning area, and its sequence is as follows (SEQ ID NO:8):

[0023] TGGGGAATATTGCACAATGGGCGGAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGTAGGGAAGAAGCGTAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCCGTGAAAGTCCGGGGCTCAACTCCGGATCTGCGGTGGGTACGGGCAGACTAGAGTGATGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCATTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCCT。

[0024] The present invention also provides a primer pair for amplifying the above-mentioned molecular marker, including:

[0025] 1) Primer pairs for amplifying specific nucleotide fragments of Scylla paramamosain cultured in Sanmen area, and their sequence information is as follows:

[0026] Forward primer F: 5′-CTCAGGATGAACGCTGACAGAATG-3′ (SEQ ID NO:9),

[0027] Reverse primer R: 5′-CACCTTAGGAACATCCCTCCAAAT-3′ (SEQ ID NO:10);

[0028] 2) Primer pairs for amplifying specific nucleotide fragments of Scylla paramamosain cultured in Ruian area, and their sequence information is as follows:

[0029] Forward primer F: 5′-CGTGGGTAACCTGCCCTAT-3′ (SEQ ID NO:11),

[0030] Reverse primer R: 5′-CGGCTATGCGTCACTTTG-3′ (SEQ ID NO:12);

[0031] 3) Primer pairs for amplifying specific nucleotide fragments of Scylla paramamosain cultured in Yunxiao area, and their sequence information is as follows:

[0032] Forward primer F: 5′-GGGAAACTGCCTGATGGA-3′ (SEQ ID NO:13),

[0033] Reverse primer R: 5′-CCTACGGTTACCTTGTTACGAC-3′ (SEQ ID NO:14);

[0034] 4) Primer pairs for amplifying specific nucleotide fragments of Scylla paramamosain cultured in Shantou area, and their sequence information is as follows:

[0035] Forward primer F: 5′-CCGCATAACATAGCACTACCG-3′ (SEQ ID NO:15),

[0036] Reverse primer R: 5′-AACCCAACATCTCACGACACGA-3′ (SEQ ID NO:16);

[0037] 5) Primer pairs for amplifying specific nucleotide fragments of Scylla paramamosain cultured in Zhuhai area, and their sequence information is as follows:

[0038] Forward primer F: 5′-GTGGGTAACCTGCCCTGTA-3′ (SEQ ID NO:17),

[0039] Downstream primer R: 5′-GCTTGCCGCCCTTTGTAT-3′ (SEQ ID NO:18);

[0040] 6) A primer pair for amplifying a specific nucleotide fragment of Scylla paramamosain cultured in Yangjiang area, and its sequence information is as follows:

[0041] Upstream primer F: 5′-GGGGATAACTACTGGAAACG-3′ (SEQ ID NO:19),

[0042] Downstream primer R: 5′-TCGCTGGCAACAAAGGAT-3′ (SEQ ID NO:20);

[0043] 7) A primer pair for amplifying a specific nucleotide fragment of Scylla paramamosain cultured in Beihai area, and its sequence information is as follows:

[0044] Upstream primer F: 5′-GCCTCTGATTTACGCTTCC-3′ (SEQ ID NO:21),

[0045] Downstream primer R: 5′-GACGACTGCCTCCTTTGC-3′ (SEQ ID NO:22);

[0046] 8) A primer pair for amplifying a specific nucleotide fragment of Scylla paramamosain cultured in Wanning area, and its sequence information is as follows:

[0047] Upstream primer F: 5′-GCCTCTGATTTACGCTTCC-3′ (SEQ ID NO:23),

[0048] Downstream primer R: 5′-GACGACTGCCTCCTTTGC-3′ (SEQ ID NO:24);

[0049] The present invention also provides a universal primer pair for amplifying the above-mentioned molecular marker, and the sequence information of its upstream and downstream primers is as follows:

[0050] Upstream primer F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO:25),

[0051] Downstream primer R: 5′-GGTTACCTTGTTACGACTT-3′ (SEQ ID NO:26);

[0052] Another aspect of the present invention also provides a probe for detecting the above-mentioned molecular marker, which includes:

[0053] 1) A probe for detecting a specific nucleotide fragment of Scylla paramamosain cultured in Sanmen area, with the sequence 5′-CACTTGACATACAACGAACTAATCAGAGATG-3′ (SEQ ID NO:27);

[0054] 2) A probe for detecting a specific nucleotide fragment of Scylla paramamosain cultured in Ruian area, with the sequence 5′-AAGGTCTTCGGATTGTAAACTTCTATCAGC-3′ (SEQ ID NO:28);

[0055] 3) A probe for detecting a specific nucleotide fragment of Scylla paramamosain cultured in Yunxiao area, with the sequence 5′-GGGAGTAAAGTTAATACCTTTGCTCATTGAC-3′ (SEQ ID NO:29);

[0056] 4) A probe for detecting a specific nucleotide fragment of Scylla paramamosain cultured in Shantou area, with the sequence 5′-GAAATGCGTAGAGATTAGGAAGAACACCAG-3′ (SEQ ID NO:30);

[0057] 5) A probe for detecting a specific nucleotide fragment of Scylla paramamosain cultured in Zhuhai area, with the sequence 5′-GTAGATATTAGGAGGAATACCAGTAGCGAAG-3′ (SEQ ID NO:31);

[0058] 6) A probe for detecting a specific nucleotide fragment of Scylla paramamosain cultured in Yangjiang area, with the sequence 5′-TTAATAACCTTATTCATTGACGTTACCCGC-3′ (SEQ ID NO:32);

[0059] 7) A probe for detecting a specific nucleotide fragment of Scylla paramamosain cultured in Beihai area, with the sequence 5′-TAGATATTTGTAGGAATGCCGATGGAGAAG-3′ (SEQ ID NO:33);

[0060] 8) A probe for detecting a specific nucleotide fragment of Scylla paramamosain cultured in Wanning area, with the sequence 5′-TAAACCTCTTTCAGTAGGGAAGAAGCGTAA-3′ (SEQ ID NO:34).

[0061] Another aspect of the present invention further provides a gene chip for detecting Scylla paramamosain from different cultured areas, and the above probes are spotted on the gene chip;

[0062] The present invention also provides a method for identifying the production areas of Scylla paramamosain, which is determined by detecting whether there are specific nucleotide fragments of the production area in the intestine of Scylla paramamosain;

[0063] More specifically, it is detected by the above-mentioned gene chip.

[0064] The present invention collects Scylla paramamosain from the main production areas of Scylla paramamosain farming in the provinces along the southeast coast of China, conducts 16S rRNA sequencing on the intestinal microbial genomes thereof, screens out specific microbial populations in each main production area through comparative analysis, comprehensively designs the characteristic DNA sequences of these populations, and finally obtains molecular markers for identifying Scylla paramamosain in each area, and develops and designs a gene chip with characteristics such as fast speed, high efficiency, and sensitivity. The origin of Sanmen Scylla paramamosain is identified by the gene chip, and non-Sanmen Scylla paramamosain is distinguished, thus solving the problem of identifying Sanmen Scylla paramamosain. Description of the Drawings

[0065] Figure 1 : Diagram of the verification result of the specific primer;

[0066] Figure 2 : Schematic diagram of the chip map;

[0067] Figure 3 : Diagram of the chip detection result. Detailed Embodiments

[0068] A gene chip, also known as a DNA chip or a biochip, refers to a technology in which a large number (usually the dot density per square centimeter is higher than 400) of probe molecules are fixed on a support and then hybridized with labeled sample molecules. By detecting the hybridization signal intensity of each probe molecule, the quantity and sequence information of the sample molecules can be obtained. Generally speaking, through microfabrication technology, tens of thousands or even millions of DNA fragments (gene probes) with specific sequences are regularly arranged and fixed on supports such as silicon wafers and glass slides of 2 cm 2 , forming a two-dimensional DNA probe array, which is very similar to the electronic chip of a computer, so it is called a gene chip.

[0069] Gene chip technology is developed based on gene probes. A gene probe is just an artificially synthesized base sequence. Some detectable substances are connected to the probe. According to the principle of base complementarity, the gene probe is used to identify specific genes in the gene mixture. It immobilizes a large number of probe molecules on a support, and then hybridizes with the labeled sample, and analyzes by detecting the intensity and distribution of the hybridization signals. By applying planar microfabrication technology and supramolecular self-assembly technology, gene chips integrate a large number of molecular detection units on the surface of a tiny solid substrate, and can simultaneously achieve high-efficiency, rapid, and low-cost detection and analysis of a large number of biological molecules such as nucleic acids and proteins. Thus, it solves the deficiencies of traditional nucleic acid blotting hybridization (such as Southern Blotting and Northern Blotting) technologies, such as complicated operations, low automation level, small number of operating sequences, and low detection efficiency.

[0070] The applicant found that there are differences in the intestinal microbial diversity of Scylla paramamosain cultured in different regions, and there are several types of intestinal microorganisms unique to the Scylla paramamosain cultured in different regions. By screening the unique intestinal microorganisms, the intestinal microbial characteristic DNA sequences for identifying Scylla paramamosain in different regions are obtained, and finally the specific molecular markers for identifying Scylla paramamosain in each region are obtained, and a chip with characteristics such as rapidity, high efficiency, and sensitivity is developed and designed. The origin of Scylla paramamosain from Sanmen is identified through chip technology, and non-Sanmen Scylla paramamosain is distinguished.

[0071] The present invention will be described in detail below in conjunction with the embodiments.

[0072] Example 1: Screening, verification of intestinal specific microorganisms of Scylla paramamosain in different regions and synthesis of nucleic acid probes

[0073] Collect 10 Scylla paramamosain samples from each main production area (Table 1), take their intestines and contents, and extract total bacterial DNA. Through 16S rRNA sequencing, the intestinal microorganisms contained in each region are analyzed, and then statistical analysis is performed on all regions, and finally the unique microorganisms of Scylla paramamosain in each region are obtained (Table 2).

[0074] Design specific amplification primers for each unique microorganism. According to the electrophoresis results of the PCR amplification products, the specific origin of Scylla paramamosain can be determined. Specific amplification primers are designed based on the V1 and V9 variable regions of the 16S rRNA gene of the unique microorganism, and a large number of experiments are carried out for screening and determination. Finally, the specific amplification primers for the unique microorganisms in each main production area are screened out. The specific sequences of the primers are shown in Table 3, and the verification results are shown in Figure 1 . The results show that the specific primers in each main production area can well identify Scylla paramamosain from each place.

[0075] Table 1: Information table of collection locations of Scylla paramamosain samples in main production areas

[0076]

[0077] Table 2: Information Table of Specific Intestinal Microorganisms of Scylla paramamosain in Each Main Production Area

[0078]

[0079]

[0080] Table 3: Information Table of Amplification Primers of Molecular Markers of Specific Intestinal Microorganisms in Each Main Production Area

[0081]

[0082] On the other hand, the present invention also designs universal primers and probes for amplifying the above molecular markers, wherein the sequence information of the universal primers is shown in Table 4, and the probe sequence information is shown in Table 5.

[0083] Table 4: Information Table of Amplification Primers for Identification Chip

[0084]

[0085] Table 5: Specific Probe Sequence Table for Different Production Areas

[0086]

[0087] Example 2: Chip Detection for Identifying Scylla paramamosain from Sanmen

[0088] 1. Extraction of DNA from Intestines and Excreta of Scylla paramamosain

[0089] Collect 10 samples of Scylla paramamosain from each main production area (Table 1), take their intestines and contents, and extract total bacterial DNA. Use DNeasy PowerSoil Kit (Catalog No.: 12888; Supplier: QIAGEN) to extract total microbial DNA, and the specific operation refers to the instruction manual.

[0090] 2. PCR Amplification, and the amplification system is shown in Table 6.

[0091] Table 6: PCR Amplification System Table

[0092]

[0093]

[0094] Table 7: PCR Reaction Program Table

[0095]

[0096] After the program ends, take out the centrifuge tube and centrifuge instantaneously to collect the solution at the bottom of the tube.

[0097] 3. Fluorescently labeled PCR products

[0098] 1) Take 20 μL of the PCR product and transfer it to a sterile and clean 0.2 mL centrifuge tube as a template.

[0099] 2) Add 5 μL of the 9N random primer solution labeled with fluorescein at a concentration of 100 μM to each sample tube, mix well by shaking, and centrifuge briefly.

[0100] 3) Place the centrifuged sample tube into a PCR instrument, denature at 95 °C for 5 min, and immediately place it on ice for 3 min.

[0101] 4) Prepare the fluorescent labeling reaction system Mix on ice according to Table 8.

[0102] Table 8: Fluorescent labeling reaction system Mix (for 1 reaction)

[0103]

[0104]

[0105] 5) Add 20 μL of the fluorescent labeling reaction system Mix to the PCR product with the random primer, mix well by shaking, and centrifuge briefly.

[0106] 6) Place the centrifuge tube into a PCR instrument, run the program set according to Table 9 for fluorescent labeling amplification. After the program ends, place the sample on ice and then proceed to the next step of chip hybridization.

[0107] Table 9: Fluorescent labeling reaction program

[0108]

[0109] 4. Chip hybridization

[0110] 1) Prepare the hybridization system

[0111] Melt the hybridization buffer at 50 °C, prepare the hybridization system according to Table 10, denature at 95 °C for 3 min, and immediately place it on ice for standby.

[0112] Table 10: Hybridization system table

[0113]

[0114] 2) Hybridization

[0115] a) Open the chip hybridization cassette, place the hybridization cassette flat on the tabletop, and add approximately 80 μL of sterilized water to the bottom groove of the hybridization cassette to prevent excessive evaporation of the hybridization solution during hybridization.

[0116] b) Place the chip face up (with the label facing the operator) between the two locating pins in the hybridization cassette.

[0117] c) Place the chip coverslip (with the convex side facing the chip), first touching the chip at the upper end and then gently covering it down.

[0118] d) Centrifuge the sample tube briefly in step 1, and slowly inject 60 μL of the denatured hybridization solution through the sample addition hole of the coverslip using a pipette. The hybridization solution will form a liquid film between the convex platform under the coverslip and the chip surface by virtue of the surface tension of the liquid. Do not vibrate the coverslip or the chip to avoid breaking the liquid film.

[0119] e) Fasten the cover plate of the hybridization cassette, ensuring that the locating pins at both ends of the groove plate are inserted into the locating pin holes at both ends of the cover plate respectively. After fastening the cover plate, clip the two metal clips onto both sides respectively. Note: The movement should be gentle and do not let the hybridization cassette have large vibrations to prevent the hybridization solution from splashing out of the dot matrix area; in addition, the metal clips need to be fully clipped to ensure the sealing of the hybridization cassette.

[0120] f) Place the hybridization cassette into the BGI BioMixerTM Ⅱ hybridization instrument (or water bath), and hybridize at 50 °C for 1 h.

[0121] Use of BGI BioMixerTM Ⅱ instrument:

[0122] ① Turn on the power supply on the back panel of the hybridization instrument and start the hybridization instrument. After the system self-check (less than 1 min) is correct, the operation interface appears on the touch screen.

[0123] ② Wait for a few seconds on the welcome interface and then automatically jump to the status interface. The protocol number and protocol name can be seen on this interface. Press the Setting button to view the specific parameters under this protocol.

[0124] ③ After selecting the protocol, press Hyb. for preheating. The system starts to run, the heating element and the fan start to work, and the hybridization cassette tray starts to rotate.

[0125] ④ After about 10 minutes, briefly turn off the Hyb. button, load the hybridization cassette and the hybridization cassette tray and then press the Hyb. button for hybridization.

[0126] 5. Chip cleaning

[0127] Use SlibeWasher TM 8-chip cleaning instrument to clean the chip.

[0128] 1) Pour 500 ml each of the pre-prepared washing solutions I and II into the reagent bottles for washing solutions I and II connected to the chip cleaning instrument respectively.

[0129] 2) Open SlibeWasherTM8 chip washer, select cleaning program 1 (the program content is 42°C, wash with washing solution I twice, 120 seconds each time, 42°C, wash with washing solution II three times, 80 seconds each time, and centrifuge to dry). Select the start button, and the cleaning program starts to run.

[0130] 3) When the washing solution I in the washer is full, take out the chip after hybridization end from the chip cassette according to the instrument prompt, quickly remove the cover glass in the cover glass collection slot of the chip washer, and then place the chip with the label facing down in the chip rack in the washer. Select the confirm button, and the chip starts to be cleaned.

[0131] 4) After the chip cleaning is completed, the washer prompts to put the chip rack into the drying chamber for centrifugal drying. After operating according to the prompt instructions, click the confirm button to start centrifuging (centrifuge at about 1500 rpm for 1 min).

[0132] 5) After centrifugation, take out the chip, and the chip can be scanned at this time.

[0133] 6. Chip scanning

[0134] Operate the LuxScan 10K-A dual-channel laser confocal scanner as follows.

[0135] 1) Start the LuxScan 10K-A application program. The program starts to search for the scanner and perform system self-check. After the system self-check is correct, the application program enters the main interface.

[0136] 2) Open the green light channel, select appropriate laser power (Laser Power) and photomultiplier gain (PMTGain) settings, and wait for the lamp to warm up.

[0137] 3) After the warm-up is completed, click the scan button, and set the fine scan area according to the pre-scanned matrix area for precise scanning.

[0138] 4) After obtaining the image, determine the origin information of the sample according to the chip atlas ( Figure 2 ). Among them, the blue circle refers to the positive control, the red circle refers to the negative control, and the yellow circles respectively represent the positions of the specific nucleotide fragment probes for culturing Scylla paramamosain in each production area.

[0139] By hybridizing the fluorescence-labeled sample with the probes on the chip, the Scylla paramamosain in different production areas can be identified. Use a laser confocal scanner to scan the chip. If bright green fluorescence appears at the corresponding position of the sample, it indicates a positive result, representing that the test sample is Scylla paramamosain from this production area; if no green fluorescence is detected at the corresponding position of the sample, it indicates a negative result for the test sample, and the sample does not belong to Scylla paramamosain from this production area.

[0140] The intestinal DNA samples from each production area were detected, and the detection results are as Figure 3 shown. As can be seen from Figure 3 , no green fluorescence was produced in the blank chip and negative control, while green fluorescence was produced at the positive control, indicating a high confidence level of the experimental results. The DNA samples of the intestinal microorganisms of Scylla paramamosain from each production area were respectively detected. The results showed that green fluorescence was only produced at the positions corresponding to each production area (positive result), while no green fluorescence was produced at the positions corresponding to other production areas (negative result), indicating that the detected sample of Scylla paramamosain belongs to this production area. The above results indicate that the gene chip provided by the present invention can effectively distinguish Scylla paramamosain cultured in the farming areas of Sanmen, Ruian, Yunxiao, Shantou, Zhuhai, Yangjiang, Beihai, and Wanning.

Claims

1. A primer set, characterized in that, The primer set is used for amplifying specific nucleotide fragments of Scylla paramamosain cultured in Sanmen, Ruian, Yunxiao, Shantou, Zhuhai, Yangjiang, Beihai, and Wanning regions; The specific nucleotide fragment of Scylla paramamosain cultured in Sanmen region has a sequence of SEQ ID NO:1; The specific nucleotide fragment of Scylla paramamosain cultured in Ruian region has a sequence of SEQ ID NO:2; The specific nucleotide fragment of Scylla paramamosain cultured in Yunxiao region has a sequence of SEQ ID NO:3; The specific nucleotide fragment of Scylla paramamosain cultured in Shantou region has a sequence of SEQ ID NO:4; The specific nucleotide fragment of Scylla paramamosain cultured in Zhuhai region has a sequence of SEQ ID NO:5; The specific nucleotide fragment of Scylla paramamosain cultured in Yangjiang region has a sequence of SEQ ID NO:6; The specific nucleotide fragment of Scylla paramamosain cultured in Beihai region has a sequence of SEQ ID NO:7; The specific nucleotide fragment of Scylla paramamosain cultured in Wanning region has a sequence of SEQ ID NO:

8.

2. The primer set according to claim 1, wherein The primer set includes: A primer pair for amplifying the specific nucleotide fragment of Scylla paramamosain cultured in Sanmen region, with the upstream primer having a sequence of SEQ ID NO:9 and the downstream primer having a sequence of SEQ ID NO:10; A primer pair for amplifying the specific nucleotide fragment of Scylla paramamosain cultured in Ruian region, with the upstream primer having a sequence of SEQ ID NO:11 and the downstream primer having a sequence of SEQ ID NO:12; A primer pair for amplifying the specific nucleotide fragment of Scylla paramamosain cultured in Yunxiao region, with the upstream primer having a sequence of SEQ ID NO:13 and the downstream primer having a sequence of SEQ ID NO:14; A primer pair for amplifying the specific nucleotide fragment of Scylla paramamosain cultured in Shantou region, with the upstream primer having a sequence of SEQ ID NO:15 and the downstream primer having a sequence of SEQ ID NO:16; A primer pair for amplifying the specific nucleotide fragment of Scylla paramamosain cultured in Zhuhai region, with the upstream primer having a sequence of SEQ ID NO:17 and the downstream primer having a sequence of SEQ ID NO:18; A primer pair for amplifying the specific nucleotide fragment of Scylla paramamosain cultured in Yangjiang region, with the upstream primer having a sequence of SEQ ID NO:19 and the downstream primer having a sequence of SEQ ID NO:20; A primer pair for amplifying the specific nucleotide fragment of Scylla paramamosain cultured in Beihai region, with the upstream primer having a sequence of SEQ ID NO:21 and the downstream primer having a sequence of SEQ ID NO:22; A primer pair for amplifying the specific nucleotide fragment of Scylla paramamosain cultured in Wanning region, with the upstream primer having a sequence of SEQ ID NO:23 and the downstream primer having a sequence of SEQ ID NO:

24.

3. The primer set according to claim 1, wherein The primer set also includes a universal amplification primer pair, with the upstream primer having a sequence of SEQ ID NO:25 and the downstream primer having a sequence of SEQ ID NO:

26.

4. A probe combination, characterized in that, The probe combination described above contains probes for detecting specific nucleotide fragments amplified by the primer set described in claim 1.

5. The probe combination according to claim 4, wherein The probe combination described above contains: A probe for detecting specific nucleotide fragments of Scylla paramamosain cultured in Sanmen area, with the sequence of SEQ ID NO:27; A probe for detecting specific nucleotide fragments of Scylla paramamosain cultured in Ruian area, with the sequence of SEQ ID NO:28; A probe for detecting specific nucleotide fragments of Scylla paramamosain cultured in Yunxiao area, with the sequence of SEQ ID NO:29; A probe for detecting specific nucleotide fragments of Scylla paramamosain cultured in Shantou area, with the sequence of SEQ ID NO:30; A probe for detecting specific nucleotide fragments of Scylla paramamosain cultured in Zhuhai area, with the sequence of SEQ ID NO:31; A probe for detecting specific nucleotide fragments of Scylla paramamosain cultured in Yangjiang area, with the sequence of SEQ ID NO:32; A probe for detecting specific nucleotide fragments of Scylla paramamosain cultured in Beihai area, with the sequence of SEQ ID NO:33; A probe for detecting specific nucleotide fragments of Scylla paramamosain cultured in Wanning area, with the sequence of SEQ ID NO:

34.

6. A gene chip for detecting Scylla paramamosain in different aquaculture regions, characterized in that, The probes in the probe combination described in claim 4 or 5 are spotted on the gene chip described above.

7. Use of the gene chip described in claim 6 in identifying Scylla paramamosain from different culture areas.

8. A method for identifying the production areas of Scylla paramamosain, characterized in that, The method described above is detected by the gene chip described in claim 6.

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