A detection method and detection kit of sea squirt environmental DNA

Abstract

The invention discloses a method for detecting environmental DNA of sea squirts and a detection kit thereof, and belongs to the field of molecular biotechnology. The invention provides primers suitable for detecting environmental DNA of at least 10 sea squirts and a multi-sample mixed efficient detection design, including: 16S-F: TTACGACCTCGATGTCG; 16S-R: YYCGTYCTAAACCCARCTC; and / or, COX1-F: CDGAYATAGCTTTTCCTCG; COX1-R: AAATACTAGAVAYYCTAGC; and / or, CYTB-F: CGTDVTATWCATRCDAAYRGDGCTAG; CYTB-R: CAAAAACTTATYTGHCYTCAAGG, and a single-tube efficient and specific amplification system using three pairs of primers at the same time, and a multi-sample mixing and splitting technical method based on primer tag sequences. Using a detection kit, amplification is performed according to a mixed PCR system, and qualitative detection of multiple sea squirts in multiple seawater environmental DNA samples can be performed simultaneously, and the detection system has high sensitivity and high accuracy.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and in particular relates to a method for detecting ascidian environmental DNA and a detection kit thereof. Background Art

[0002] Ascidian is a typical fouling organism and invasive species, and typical species include Ciona, Ascidian, Salmonella, Leather, Ascidian and Ascidian. These ascidian can grow rapidly and quickly replace other species in their native habitats or invasive areas. They are known fouling organisms of ships and docks, which seriously affect shipping. Studies have also shown that the invasion of Ciona can also have a serious impact on aquaculture. They will occupy the culture ropes or culture cages of aquatic organisms such as scallops and oysters, compete for oxygen and bait, pollute aquaculture equipment and ships, and also retain and transmit the living cells and cysts of toxic phytoplankton, causing a large number of deaths of aquaculture organisms, thereby directly or indirectly causing huge economic losses. Therefore, the safety risks such as biological invasion caused by ascidian are increasingly concerned, and it is of great significance to establish a fast and accurate detection method for preventing and controlling the invasion of ascidian, but it is difficult to obtain sufficient data by relying solely on traditional survey methods, and the accuracy of the survey needs to be strengthened.

[0003] Environmental DNA (eDNA) detection is a species detection method that has emerged in recent years. It extracts environmental DNA by collecting water samples and uses specific molecular markers to detect aquatic organisms. It has the advantages of being non-invasive to the ecological environment, simple and efficient sampling, high detection sensitivity, and accurate species identification. In the process of using environmental DNA detection methods to detect target species, if only a single target is used, it will be difficult to define closely related species. Multi-target detection of target species can not only improve the detection rate of target species, but also improve interspecies resolution. It is of great significance to research and develop multiple detection targets for target species, design short fragment amplification primers, screen molecular markers that can be stably amplified, design efficient multi-target primer mixed PCR systems and multi-sample mixed amplicon library establishment methods, design rapid detection kits, and further research and develop detection methods suitable for sea squirt environmental DNA. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a method for detecting environmental DNA of sea squirts and a detection kit thereof. The present invention designs three detection targets for sea squirts and three corresponding pairs of primers, designs an efficient multi-target primer mixed PCR system, designs a method for establishing a multi-sample mixed amplicon library, and designs a rapid detection kit. It can quickly and accurately detect invasive sea squirts, so as to formulate corresponding prevention policies in a timely manner and reduce the harm caused by invasive sea squirts.

[0005] To achieve the above object, the present invention provides a primer for detecting environmental DNA of ascidian, comprising:

[0006] 16S-F:TTACGACCTCGATGTCG;

[0007] 16S-R:YYCGTYCTAAACCCARCTC; and / or,

[0008] COX1-F: CDGAYATAGCTTTTCCTCG;

[0009] COX1-R:AAATACTAGAVAYYCTAGC; and / or,

[0010] CYTB-F:CGTDVTATWCATRCDAAYRGDGCTAG;

[0011] CYTB-R:CAAAAACTTATYTGHCYTCAAGG.

[0012] Furthermore, the 5' end of the primer is also marked with a sample tag, and the sample tag is a marker sequence for identifying different samples to be tested.

[0013] Furthermore, the marker sequence includes:

[0014]

[0015] The present invention also provides a use of the above-mentioned primers for detecting environmental DNA of ascidian in detecting ascidian.

[0016] The present invention also provides a method for detecting ascidian, comprising the following steps:

[0017] Using the environmental DNA sample to be tested as a template, PCR amplification is performed using the above primers;

[0018] The amplified product is analyzed for the presence of target DNA fragments to determine whether ascidians are present in the environmental DNA sample to be tested.

[0019] Furthermore, the target DNA fragment is a specific DNA fragment of the ascidian.

[0020] Furthermore, the environmental DNA sample to be tested is seawater.

[0021] Furthermore, the method also includes the step of enriching and / or mixing the environmental DNA sample to be tested.

[0022] Furthermore, the reaction system of PCR amplification includes: 100 ng of the environmental DNA sample to be tested, 5.5 μL of premixed primers, 1 μL of DNA polymerase, 25 μL of Buffer Mix, and sterile enzyme-free water to make up to 50 μL;

[0023] The premixed primers include 2.5 μL of COX1 primer pair, 0.5 μL of 16S primer pair, and 2.5 μL of CYTB primer pair; the final reaction concentration of the DNA polymerase is 1 U;

[0024] The reaction conditions of the PCR amplification are: 94°C for 1 min, 98°C for 10 s, 45°C for 15 s, 68°C for 15 s, 20 cycles, 98°C for 10 s, 50°C for 15 s, 68°C for 15 s, 15 cycles, 68°C for 5 min, and storage at 12°C.

[0025] Furthermore, the method for analyzing whether the amplified product contains the target DNA fragment includes: agarose gel electrophoresis and / or fluorescent dye method.

[0026] The present invention also provides a kit for detecting environmental DNA of ascidian, comprising the above-mentioned primers for detecting environmental DNA of ascidian; and also comprising a 48-well plate, PCR master mix (2X), DNA clean beads, End-repair and A-tailing Reaction Mix, End-repair and A-tailing Enzyme Mix, Adapter for Illumina, Adapter Dilution Buffer, Adapter-ligation Buffer, Adapter-ligation Enzyme, Library Amplification Super Mix (2X), Universal Primer Mix for Illumina, Library Elution Buffer and Nuclease-free Water.

[0027] Compared with the prior art, the present invention has the following advantages and technical effects:

[0028] The present invention provides primers for detecting environmental DNA of ascidian, a multi-target mixed PCR system, a multi-sample mixed amplicon library construction method, and an ascidian environmental DNA detection kit. The environmental DNA is detected by using the technical process, and the ascidian species present in the seawater environmental DNA sample can be detected. The method is simple and fast, and the detection sensitivity and accuracy are high. The present invention provides a reliable theoretical and technical basis for the prevention and control of ascidian invasive organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 The ascidian species and the corresponding COX1 read percentages in the 9 environmental DNA samples were compared;

[0031] Figure 2 The ascidian species and the corresponding COX1 read numbers mapped in the 9 environmental DNA samples;

[0032] Figure 3 The ascidian species and the corresponding CYTB read percentages in the 9 environmental DNA samples were compared;

[0033] Figure 4 The ascidian species and the corresponding CYTB read numbers mapped in the 9 environmental DNA samples;

[0034] Figure 5 The ascidian species and the corresponding 16S read percentages in the 9 environmental DNA samples were compared;

[0035] Figure 6 The ascidian species and the corresponding 16S read numbers in the 9 environmental DNA samples were compared;

[0036] Figure 7 is the percentage of reads of the aligned 16S amplicon, CYTB amplicon, and COX1 amplicon;

[0037] Figure 8 The types of ascidian species mapped in the 9 environmental DNA samples and the corresponding percentage of reads;

[0038] Fig. 9 The ascidian species and corresponding read numbers mapped in 9 environmental DNA samples. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] Example 1

[0045] 1. Amplification and quality control of multi-target environmental DNA (eDNA) target fragments

[0046] 1. Detection of target

[0047] The target genes detected are mitochondrial genes 16S, COX1 and CYTB.

[0048] 2. Primer Design

[0049] 16S forward primer

[0050] TTACGACCTCGATGTCG;

[0051] 16S reverse primer

[0052] YYCGTYCTAAACCCARCTC;

[0053] COX1 forward primer

[0054] CDGAYATAGCTTTTCCTCG;

[0055] COX1 reverse primer

[0056] AAATACTAGAVAYYCTAGC;

[0057] CYTB forward primer

[0058] CGTDVTATWCATRCDAAYRGDGCTAG;

[0059] CYTB reverse primer

[0060] CAAAAACTTATYTGHCYTCAAGG.

[0061] When designing primers, the mitochondrial gene sequences of four species of ascidians were used as reference. The target DNA sequences targeted by the three pairs of primers are as follows:

[0062] 16S ascidian:

[0063] TTACGACCTCGATGTCGACTTGTACTTTATTTAAATTTGCAGTAAAATTTAATTTATGTTTGTTCACCATTTAATAGTGTACGTGAGTTGGGTTTAGAACGGG

[0064] 16S Ciona:

[0065] TTACGACCTCGATGTCGGTTTACTTTAACTAATTCTTGTAGAAGAGAATAAAGTTTGTTTGCTAACCAAATTATCAAGTACATGAGCTGGGTTAAGGACGAA

[0066] 16S Salmonella:

[0067] TTACGACCTCGATGTCGGTTTTATTTTATCTATTTCTCGCAAAAGAGAAAAAAGTTTGTTTGCTAACCAATTAAAAAAATACATGAGCTTGGGTTAAGGACGAA

[0068] 16S true ascidian:

[0069] TTACGACCTCGATGTCGACTTAGGTTGTTAGCTACGCGCAGAAGTGTAGCTTGTTTGTTTGTTCACCAATTAAGAACCTACGTGAGTTGGGTTTAGAAACGG

[0070] Ciona savignyi:

[0071] CGTTCTATTCATATGAATAGAGCTAGTTTTTTTTTGTTTTGTATTTTTATGCATTTAGGCCGAGGGCTTTATTATAAATCTTTTATAAATATCCATACTTGGGGGGTGAGAGTAATTTTATTAATTTTAAGTATGATTACTTCTTTTCTTAGGTATGTTTTACCTTGAAGACAAATGAGTTTTTG

[0072] Ciona intestinalis:

[0073] CGTTATATACATGCTAACAGAGCTAGTTTTTTTTTAGCATTTATTTTTATACACATTAGACGAAATCTTTATTATAAAACTTATTATCGAAAAAGAGCTTGATTAGTAAGAATTGTTTTATTAATTCTTTCAATATTAACTGCTTTTTTAAGATATGTTTTACCTTGAAGACAAATAAGTTTTTG

[0074] Ciona savignyi:

[0075] CGTTATATACATGCAAATGGTGCTAGTTTTTTTTTGAGATTGATTTTTTTACATATTAGACGAAATGTTTATTATAAAACTTATTATCGTAAGGGGGCTTGGATTATTGGTATCTTACTTTTAGTAGTATCCATGTTAACTGCTTTTTTAGGTTATGTTTTACCGTGAGGTCAGATAAGTTTTTG

[0076] Ciona robusta:

[0077] CGTAGTGTTCATGCTAATGGGGCTAGTTTTTTTATATTTGGTTTATTTATACATGTAGGTCGTGGTTTGTATTATAAGTCGTATTTGGATTTTCGAGTTTGGTATGTGGGGGTAATTTTGTTGGTTGTTAGTATATTAACTTCTTTTTTTAGGTATGTTTTGCCTTGAGGGCAGATAAGTTTTTG

[0078] Ciona savignyi:

[0079] GACATAGCTTTTCCACGTCTAAATAATATGAGTTTTTGGTTATTGCCTCCGGCGGTAGTCCTTTTATTAGTAAGCTCCTTTATTGAGAGTGGAGTAGGAACTAGGTGGACTGTGTACCCTCCTTTAGCTAGGAACTTGGCCCATTCAAGAGGTGCTCTTGATTGTGCTATTTTTTCTTTACATTTGGCGGGGATCTCCAGTATTT

[0080] Ciona intestinalis:

[0081] CTGATATAGCTTTTCCTCGGATAAATAATATGAGTTTTTGACTATTACCTCCTTCTTTTTTTTTATTATTAAAAAGTAGATTCTCAGGTTCAGCTAGAGTAAGAACTGGGTGAACAGTTTACCCTCCTTTATCTGCAAATATCAGACACTCTAATACTAGAGTTGATATAGCTATTTTTTCTTTACATTTAGCTAGAGTTTCTAGTATTT

[0082] Ciona savignyi:

[0083] CGGATATAGCGTTTCCTCGAATAAATAATATAAGCTTTTGATTATTACCTCCTGCTTTTTTTATGTTATTAAAAAGTAGTTTTGCAGGAGCATCGAGAGTAAGAACTGGATGGACAGTTTATCCTCCTTTGTCTGCTACGGTAAGACATGCTCATTCTAGAGTAGATATAGCTATTTTTTCATTACATTTAGCTAGGGTTTCTAGTATTT

[0084] Ciona robusta:

[0085] CAGATATGGCTTTTCCTCGATTAAATAATATGAGTTTTTGGTTATTGCCTCCTGCGTTATTTATGCTATTGTTATCTTCAGTAATTGAGAGTGGTGTAAGAACAGGTTGGACTGTTTATCCACCTTTATCAAGGAATTTAGCTCATTCTGGGCCGGCACTTGATTGTGCTATTTTTTCTTTGCATTTAGCTAGAGTGTCTAGTATTT

[0086] 3. Sample label

[0087]

[0088]

[0089] 4. Primers for adding sample labels: 1-16S-F TCTTCTGCCTTACGACCTCGATGTCG 1-16S-R TCTGACCTTYYCGTYCTAAACCCARCTC 2-16S-F TCTGTCTCTTTACGACCTCGATGTCG 2-16S-R TCTTCATAAYYCGTYCTAAACCCARCTC 3-16S-F TCTATATTGTTACGACCTCGATGTCG 3-16S-R TCTCAAGAGYYCGTYCTAAACCCARCTC 4-16S-F TCTTGGAAGTTACGACCTCGATGTCG 4-16S-R TCTCGATCAYYCGTYCTAAACCCARCTC 5-16S-F TCTTCTAGTTTACGACCTCGATGTCG 5-16S-R TCTTTGATTYYCGTYCTAAACCCARCTC 6-16S-F TCTAGAGTATTACGACCTCGATGTCG 6-16S-R TCTTCCGAGYYCGTYCTAAACCCARCTC 7-16S-F TCTGGCCAATTACGACCTCGATGTCG 7-16S-R TCTCCTGAAYYCGTYCTAAACCCARCTC 8-16S-F TCTTATCTCTTACGACCTCGATGTCG 8-16S-R TCTATTCTTYYCGTYCTAAACCCARCTC 9-16S-F TCTTTATGCTTACGACCTCGATGTCG

[0090] 9-16S-R TCTGCGACTYYCGTYCTAAACCCARCTC

[0091] 10-16S-F TCTAGTTGGTTACGACCTCGATGTCG

[0092] 10-16S-R TCTGGCTTCYYCGTYCTAAACCCARCTC

[0093] 11-16S-F TCTGTCAAGTTACGACCTCGATGTCG

[0094] 11-16S-R TCTAATACGYYCGTYCTAAACCCARCTC

[0095] 12-16S-F TCTCAGCAATTACGACCTCGATGTCG

[0096] 12-16S-R TCTTACGGTYYCGTYCTAAACCCARCTC

[0097] 13-16S-F TCTTCGCCGTTACGACCTCGATGTCG

[0098] 13-16S-R TCTACCGTCYYCGTYCTAAACCCARCTC

[0099] 14-16S-F TCTCTAAGATTACGACCTCGATGTCG

[0100] 14-16S-R TCTAGAAGCYYCGTYCTAAACCCARCTC

[0101] 15-16S-F TCTCCGCTTTTACGACCTCGATGTCG

[0102] 15-16S-R TCTCATAGCYYCGTYCTAAACCCARCTC

[0103] 16-16S-F TCTAAGTTATTACGACCTCGATGTCG

[0104] 16-16S-R TCTAGGCTCYYCGTYCTAAACCCARCTC

[0105] 17-16S-F TCTGGTACCTTACGACCTCGATGTCG

[0106] 17-16S-R TCTCTGCGGYYCGTYCTAAACCCARCTC

[0107] 18-16S-F TCTCCAGGTTTACGACCTCGATGTCG

[0108] 18-16S-R TCTCTCGGCYYCGTYCTAAACCCARCTC

[0109] 19-16S-F TCTAATCGATTACGACCTCGATGTCG

[0110] 19-16S-R TCTGATTAGYYCGTYCTAAACCCARCTC

[0111] 20-16S-F TCTAACGCATTACGACCTCGATGTCG

[0112] 20-16S-R TCTAGATATYYCGTYCTAAACCCARCTC

[0113] 21-16S-F TCTCTAGACTTACGACCTCGATGTCG

[0114] 21-16S-R TCTGGCATCYYCGTYCTAAACCCARCTC

[0115] 22-16S-F TCTCATTAGTTACGACCTCGATGTCG

[0116] 22-16S-R TCTCGCTAGYYCGTYCTAAACCCARCTC

[0117] 23-16S-F TCTAGCCTGTTACGACCTCGATGTCG

[0118] 23-16S-R TCTCCATAGYYCGTYCTAAACCCARCTC

[0119] 24-16S-F TCTGGTCTGTTACGACCTCGATGTCG

[0120] 24-16S-R TCTAATGTCYYCGTYCTAAACCCARCTC

[0121] 1-COX1-F TCTTCTGCCCDGAYATAGCTTTTCCTCG

[0122] 1-COX1-R TCTGACCTTAAATACTAGAVAYYCTAGC

[0123] 2-COX1-F TCTGTCTCTCDGAYATAGCTTTTCCTCG

[0124] 2-COX1-R TCTTCATAAAAATACTAGAVAYYCTAGC

[0125] 3-COX1-F TCTATATTGCDGAYATAGCTTTTCCTCG

[0126] 3-COX1-R TCTCAAGAGAAATACTAGAVAYYCTAGC

[0127] 4-COX1-F TCTTGGAAGCDGAYATAGCTTTTCCTCG

[0128] 4-COX1-R TCTCGATCAAAATACTAGAVAYYCTAGC

[0129] 5-COX1-F TCTTCTAGTCDGAYATAGCTTTTCCTCG

[0130] 5-COX1-R TCTTTGATTAAATACTAGAVAYYCTAGC

[0131] 6-COX1-F TCTAGAGTACDGAYATAGCTTTTCCTCG

[0132] 6-COX1-R TCTTCCGAGAAATACTAGAVAYYCTAGC

[0133] 7-COX1-F TCTGGCCAACDGAYATAGCTTTTCCTCG

[0134] 7-COX1-R TCTCCTGAAAAATACTAGAVAYYCTAGC

[0135] 8-COX1-F TCTTATCTCCDGAYATAGCTTTTCCTCG

[0136] 8-COX1-R TCTATTCTTAAATACTAGAVAYYCTAGC

[0137] 9-COX1-F TCTTTATGCCDGAYATAGCTTTTCCTCG

[0138] 9-COX1-R TCTGCGACTAAATACTAGAVAYYCTAGC

[0139] 10-COX1-F TCTAGTTGGCDGAYATAGCTTTTCCTCG

[0140] 10-COX1-R TCTGGCTTCAAATACTAGAVAYYCTAGC

[0141] 11-COX1-F TCTGTCAAGCDGAYATAGCTTTTCCTCG

[0142] 11-COX1-R TCTAATACGAAATACTAGAVAYYCTAGC

[0143] 12-COX1-F TCTCAGCAACDGAYATAGCTTTTCCTCG

[0144] 12-COX1-R TCTTACGGTAAATACTAGAVAYYCTAGC

[0145] 13-COX1-F TCTTCGCCGCDGAYATAGCTTTTCCTCG

[0146] 13-COX1-R TCTACCGTCAAATACTAGAVAYYCTAGC

[0147] 14-COX1-F TCTCTAAGACDGAYATAGCTTTTCCTCG

[0148] 14-COX1-R TCTAGAAGCAAATACTAGAVAYYCTAGC

[0149] 15-COX1-F TCTCCGCTTCDGAYATAGCTTTTCCTCG

[0150] 15-COX1-R TCTCATAGCAAATACTAGAVAYYCTAGC

[0151] 16-COX1-F TCTAAGTTACDGAYATAGCTTTTCCTCG

[0152] 16-COX1-R TCTAGGCTCAAATACTAGAVAYYCTAGC

[0153] 17-COX1-F TCTGGTACCCDGAYATAGCTTTTCCTCG

[0154] 17-COX1-R TCTCTGCGGAAATACTAGAVAYYCTAGC

[0155] 18-COX1-F TCTCCAGGTCDGAYATAGCTTTTCCTCG

[0156] 18-COX1-R TCTCTCGGCAAATACTAGAVAYYCTAGC

[0157] 19-COX1-F TCTAATCGACDGAYATAGCTTTTCCTCG

[0158] 19-COX1-R TCTGATTAGAAATACTAGAVAYYCTAGC

[0159] 20-COX1-F TCTAACGCACDGAYATAGCTTTTCCTCG

[0160] 20-COX1-R TCTAGATATAAATACTAGAVAYYCTAGC

[0161] 21-COX1-F TCTCTAGACCDGAYATAGCTTTTCCTCG

[0162] 21-COX1-R TCTGGCATCAAATACTAGAVAYYCTAGC

[0163] 22-COX1-F TCTCATTAGCDGAYATAGCTTTTCCTCG

[0164] 22-COX1-R TCTCGCTAGAAATACTAGAVAYYCTAGC

[0165] 23-COX1-F TCTAGCCTGCDGAYATAGCTTTTCCTCG

[0166] 23-COX1-R TCTCCATAGAAATACTAGAVAYYCTAGC

[0167] 24-COX1-F TCTGGTCTGCDGAYATAGCTTTTCCTCG

[0168] 24-COX1-R TCTAATGTCAAATACTAGAVAYYCTAGC

[0169] 1-CYTB-F TCTTCTGCCCGTDVTATWCATRCDAAYRGDGCTAG

[0170] 1-CYTB-R TCTGACCTTCAAAAACTTATYTGHCYTCAAGG

[0171] 2-CYTB-F TCTGTCTCTCGTDVTATWCATRCDAAYRGDGCTAG

[0172] 2-CYTB-R TCTTCATAACAAAAACTTATYTGHCYTCAAGG

[0173] 3-CYTB-F TCTATATTGCGTDVTATWCATRCDAAYRGDGCTAG

[0174] 3-CYTB-R TCTCAAGAGCAAAAACTTATYTGHCYTCAAGG

[0175] 4-CYTB-F TCTTGGAAGCGTDVTATWCATRCDAAYRGDGCTAG

[0176] 4-CYTB-R TCTCGATCACAAAAACTTATYTGHCYTCAAGG

[0177] 5-CYTB-F TCTTCTAGTCGTDVTATWCATRCDAAYRGDGCTAG

[0178] 5-CYTB-R TCTTTGATTCAAAAACTTATYTGHCYTCAAGG

[0179] 6-CYTB-F TCTAGAGTACGTDVTATWCATRCDAAYRGDGCTAG

[0180] 6-CYTB-R TCTTCCGAGCAAAAACTTATYTGHCYTCAAGG

[0181] 7-CYTB-F TCTGGCCAACGTDVTATWCATRCDAAYRGDGCTAG

[0182] 7-CYTB-R TCTCCTGAACAAAAACTTATYTGHCYTCAAGG

[0183] 8-CYTB-F TCTTATCTCCGTDVTATWCATRCDAAYRGDGCTAG

[0184] 8-CYTB-R TCTATTCTTCAAAAACTTATYTGHCYTCAAGG

[0185] 9-CYTB-F TCTTTATGCCGTDVTATWCATRCDAAYRGDGCTAG

[0186] 9-CYTB-R TCTGCGACTCAAAAACTTATYTGHCYTCAAGG

[0187] 10-CYTB-F TCTAGTTGGCGTDVTATWCATRCDAAYRGDGCTAG

[0188] 10-CYTB-R TCTGGCTTCCAAAAACTTATYTGHCYTCAAGG

[0189] 11-CYTB-F TCTGTCAAGCGTDVTATWCATRCDAAYRGDGCTAG

[0190] 11-CYTB-R TCTAATACGCAAAAACTTATYTGHCYTCAAGG

[0191] 12-CYTB-F TCTCAGCAACGTDVTATWCATRCDAAYRGDGCTAG

[0192] 12-CYTB-R TCTTACGGTCAAAAACTTATYTGHCYTCAAGG

[0193] 13-CYTB-F TCTTCGCCGCGTDVTATWCATRCDAAYRGDGCTAG

[0194] 13-CYTB-R TCTACCGTCCAAAAACTTATYTGHCYTCAAGG

[0195] 14-CYTB-F TCTCTAAGACGTDVTATWCATRCDAAYRGDGCTAG

[0196] 14-CYTB-R TCTAGAAGCCAAAAACTTATYTGHCYTCAAGG

[0197] 15-CYTB-F TCTCCGCTTCGTDVTATWCATRCDAAYRGDGCTAG

[0198] 15-CYTB-R TCTCATAGCCAAAAACTTATYTGHCYTCAAGG

[0199] 16-CYTB-F TCTAAGTTACGTDVTATWCATRCDAAYRGDGCTAG

[0200] 16-CYTB-R TCTAGGCTCCAAAAACTTATYTGHCYTCAAGG

[0201] 17-CYTB-F TCTGGTACCCGTDVTATWCATRCDAAYRGDGCTAG

[0202] 17-CYTB-R TCTCTGCGGCAAAAACTTATYTGHCYTCAAGG

[0203] 18-CYTB-F TCTCCAGGTCGTDVTATWCATRCDAAYRGDGCTAG

[0204] 18-CYTB-R TCTCTCGGCCAAAAACTTATYTGHCYTCAAGG

[0205] 19-CYTB-F TCTAATCGACGTDVTATWCATRCDAAYRGDGCTAG

[0206] 19-CYTB-R TCTGATTAGCAAAAACTTATYTGHCYTCAAGG

[0207] 20-CYTB-F TCTAACGCACGTDVTATWCATRCDAAYRGDGCTAG

[0208] 20-CYTB-R TCTAGATATCAAAAACTTATYTGHCYTCAAGG

[0209] 21-CYTB-F TCTCTAGACCGTDVTATWCATRCDAAYRGDGCTAG

[0210] 21-CYTB-R TCTGGCATCCAAAAACTTATYTGHCYTCAAGG

[0211] 22-CYTB-FTCTCATTAGCGTDVTATWCATRCDAAYRGDGCTAG

[0212] 22-CYTB-RTCCTCGCTAGCAAAAACTTATYTGHCYTCAAGG

[0213] 23-CYTB-F TCTAGCCTGCGTDVTATWCATRCDAAYRGDGCTAG23-CYTB-R TCTCCATAGCAAAAACTTATYTGHCYTCAAGG 24-CYTB-F TCTGGTCTGCGTDVTATWCATRCDAAYRGDGCTAG 24-CYTB-RTCTAATGTCCAAAAACTTATYTGHCYTCAAGG

[0214] 5. Environmental DNA samples

[0215] The method of the present invention was used to test the 9 environmental DNA samples in the table below.

[0216] 9 environmental DNA samples

[0217]

[0218] 6. PCR amplification of multi-target environmental DNA target fragments

[0219] 6.1 PCR system and program settings are shown in the following table.

[0220]

[0221]

[0222]

[0223] 7. Gel electrophoresis detection

[0224] The PCR amplification products were subjected to 1.0% agarose gel electrophoresis, and the electrophoresis results were photographed and stored in a gel imaging system.

[0225] 8. Sample Mixing

[0226] Take 5 μL of the PCR amplification products of each of the 9 samples and mix them.

[0227] 9. After the amplicon is mixed, the sample needs to be purified. Solid-phase reversible immobilization (SPRI) paramagnetic beads (referred to as "magnetic beads") can be used to recover all DNA fragments in the sample. The method is as follows:

[0228] 9.1. Take out the DNA fragment screening reagent from 4°C half an hour in advance and let its temperature equilibrate to room temperature;

[0229] 9.2. Add 50μL sample to the centrifuge tube, then add the magnetic bead suspension, mix well by pipetting 10 times, and let stand at room temperature for 5 minutes: Place the centrifuge tube on the magnetic separator until the solution becomes clear, and remove the supernatant with a pipette; Note: Sample volume x ratio = amount of magnetic bead suspension added, such as 50μL sample x 0.6 = 30μL magnetic bead suspension;

[0230] 9.3. Keep the centrifuge tube on the magnetic separator, add 200 μL 80% ethanol, let it stand at room temperature for 30 seconds, and then remove the supernatant with a pipette: Repeat this step once;

[0231] 9.4. Keep the centrifuge tube on the magnetic separator and air dry until the surface of the magnetic beads has no obvious gloss;

[0232] 9.5. Add 30-40 μL of sterile enzyme-free water to the centrifuge tube, pipette repeatedly 10 times to mix the magnetic beads and the solution thoroughly, and let the reaction tube stand at room temperature for 3-5 minutes; then place the centrifuge tube in a magnetic separator until the solution becomes clear, transfer the supernatant to a new centrifuge tube, which can be used directly for subsequent research or placed in a -20°C refrigerator for long-term storage.

[0233] 10. The DNA concentration of the purified product was measured using a fluorescent dye method based on specific recognition of dsDNA. The concentrations are shown in the following table.

[0234]

[0235] 11. Construction of mixed sample library of multi-target environmental DNA amplicons

[0236] 11.1. End repair and A addition reaction

[0237] 11.1.1. Place a sterile PCR tube on ice and add the system shown in the table below.

[0238] Element volume Starting sample (1ng-1ug of dsDNA) Variable (10-25μL) Sterile enzyme-free water Variable End-repair and A-tailing Reaction Mix 10μL End-repair and A-tailing Enzyme Mix 5μL Total volume 60μL

[0239] 11.1.2. Mix by pipetting.

[0240] 11.1.3. Place the reaction tube in the PCR instrument and perform the following incubation steps (heat cover temperature ≥ 80°C):

[0241] 28℃ 15min

[0242] 68℃ 15min

[0243] ≤10℃ hold.

[0244] 11.2. Connector connection

[0245] 11.2.1 Thaw the Adapter on ice and dilute it with sterile enzyme-free water. The conditions are shown in the table below.

[0246] Starting sample size Adapter dilution factor Adapter concentration after dilution 100ng≤x≤1ug No dilution 16μL 25ng≤x<100ng Dilute 2 times 8μL 5ng≤x<25ng Dilute 10 times 1.6μL 1ng≤x<5ng Dilute 25 times 0.6μL

[0247] 11.2.2 Place the PCR tube where the previous reaction is completed on ice and add the following system in sequence.

[0248] Element volume Adapter with appropriate concentration 5μL Adapter Dilution Buffer 30μL Adapter-ligation Buffer 5μL Total volume 100μL

[0249] 11.2.3 Mix by pipetting.

[0250] 11.2.4 Place the reaction tube in a PCR instrument and incubate at 20°C for 15 minutes (the lid is not heated). Purify the ligated product immediately.

[0251] 11.3. Purification of adapter ligation products

[0252] Use magnetic beads to recover and purify all DNA fragments larger than 50 bp in the sample.

[0253] 11.3.1 Take out the DNA fragment screening reagent from 4°C half an hour in advance and allow its temperature to equilibrate to room temperature.

[0254] 11.3.2 Add 100 μL of sample to a centrifuge tube, then add the magnetic bead suspension, mix by pipetting 10 times, and let stand at room temperature for 5 min. Place the centrifuge tube on a magnetic separator until the solution becomes clear, and remove the supernatant with a pipette.

[0255] 11.3.3 Keep the centrifuge tube on the magnetic separator, add 200 μL 80% ethanol, let it stand at room temperature for 30 seconds, and then remove the supernatant with a pipette; repeat this step once.

[0256] 11.3.4 Keep the centrifuge tube on the magnetic separator and air-dry until the surface of the magnetic beads has no obvious gloss.

[0257] 11.3.5 Add 20 μL of sterile enzyme-free water to the centrifuge tube, pipette repeatedly 10 times to mix the magnetic beads and the solution thoroughly, and let the reaction tube stand at room temperature for 3 to 5 minutes; then place the centrifuge tube in a magnetic separator until the solution becomes clear, transfer the supernatant to a new centrifuge tube, and it can be directly used for subsequent library amplification.

[0258] 11.4 Library Amplification

[0259] 11.4.1 Place a sterile PCR tube on ice and add the following system.

[0260]

[0261] 11.4.2 Mix by pipetting.

[0262] 11.4.3 Amplify in a PCR instrument with the following program: 98°C for 3 min, 98°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec, 2 cycles, 72°C for 3 min, and store at 10°C.

[0263] 11.5. Purification of library amplification products

[0264] Use magnetic beads to recover and purify all DNA fragments larger than 50 bp in the sample. The specific method is the same as 11.3.

[0265] 11.6. Quality Control after Library Construction

[0266] 11.6.1 After the library is built, the DNA concentration of the samples is measured using a fluorescent dye method based on the specific recognition of dsDNA. The concentration determination is shown in the following table.

[0267] Sample name dsDNA fluorescent dye concentration determination Mixed amplicon library samples 25.32 ng / μL

[0268] It can be seen that the concentration of the mixed amplicon library construction sample needs to reach the minimum concentration required by the sequencing company.

[0269] 11.7 Off-line Data Analysis

[0270] 11.7.1 Use the double-end sequencing splicing software PANDAseq to splice the two sequencing files R1 and R2 into a complete fastq file.

[0271] 11.7.2 Use the awk tool to extract the reads of different sampling points from the complete fastq obtained in the previous step according to the tag sequence.

[0272] 11.7.3 After the sequencing data from different sampling points were split and named separately, the mitochondrial sequences, COX1 sequences, 16S sequences, and CYTB sequences of 114 invasive species of ascidians were collected and sorted out as reference databases.

[0273] 11.7.4 Use blastn software for comparison.

[0274] 11.7.5 Use the cut tool to count the different invasive species of ascidian and the corresponding read numbers in the alignment results.

[0275] 11.7.6 Statistical Results

[0276] 11.7.6.1 Using the COX1 gene sequences of 66 species of ascidian as a reference database, blastn was used to compare the species of ascidian compared to the number of corresponding reads and their percentages, such as Figure 1 and Figure 2 shown.

[0277] 11.7.6.2 Using the CYTB gene sequences of 22 species of ascidian as a reference database, blastn was used to compare the species of ascidian compared to the number of corresponding reads and their percentages, such as Figure 3 and Figure 4 shown.

[0278] 11.7.6.3 Using the 16S gene sequences of 19 species of ascidian as a reference database, blastn was used to compare the species of ascidian compared to the number of corresponding reads and their percentages, such as Figure 5 and Figure 6 shown.

[0279] 11.7.6.4 Count the number of reads and their proportions obtained by comparing the three databases, such as Figure 7 shown.

[0280] 11.7.6.5 Based on the comparison results of the three databases, the number of matched ascidian species and the corresponding read segments and their percentages were counted, such as Figure 8 and Fig. 9 shown.

[0281] In summary, judging from the number of 16S amplicon reads and the corresponding ascidian species at the nine sampling sites, the detected ascidian species include the ascidian species witnessed at each sampling site, and the number of 16S amplicon reads of the witnessed ascidian species is significantly higher than that of the unobserved ascidian species. The COX1 and CYTB amplicons did not fully detect all the witnessed ascidian species, but other ascidian species that were not detected by the 16S amplicon were detected. By integrating the ascidian species detected by the three amplicons, the ascidian species present in the environmental DNA samples can be fully, comprehensively, efficiently and quickly detected.

[0282] Example 2

[0283] A kit for detecting environmental DNA of sea squirts, comprising kit 1 and kit 2:

[0284] Among them, the kit 1 (stored at 4°C) includes:

[0285] 1. 48-well plate (including the above-mentioned primers for the detection of ascidian environmental DNA): Each well contains 3 pairs of primer mixed powders, and the primers in each well have different index sequences; the user needs to use 5.5 μL of sterile enzyme-free water to dissolve the primer powder in the 48-well plate and prepare it on the spot when using;

[0286] 2. DNAcleanbeads: used to purify samples after PCR amplification and during library construction;

[0287] 3.Nuclease-free Water;

[0288] Kit 2 (stored at -20°C) includes:

[0289] 1. PCRmastermix (2X): used for PCR amplification reaction in the above process;

[0290] 2. End-repair and A-tailing Reaction Mix, End-repair and A-tailing Enzyme Mix: used for end repair and A-tailing reaction in the above library construction process;

[0291] 3. Adapter for Illumina, Adapter Dilution Buffer, Adapter-ligation Buffer, Adapter-ligation Enzyme: used for adapter ligation reaction in the above library construction process;

[0292] 4. Library Amplification SuperMix (2X), Universal Primer Mix for Illumina: used for library amplification reaction in the above library construction process.

[0293] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for detecting fouling and invasion of ascidian, characterized in that The following steps are involved: Environmental DNA samples from seawater were used as templates, and primers optimized for the detection target and with sample labels were used for second-generation high-throughput sequencing; In one round of PCR reaction, three highly variable target regions between species can be amplified and double-end sample labeled simultaneously; The mixed sample library was constructed using the end-filling plus sequencing adapter scheme; Using the reference database of major fouling and invasive ascidians along the coast of China and data analysis tools, the species and relative abundance of fouling and invasive ascidians in the environmental DNA samples were detected based on the results of second-generation high-throughput sequencing; The primers optimized for the detection target and with sample labels include: Among them, base Y is C or T, base R is A or G, base D is T or A or G, base V is G or A or C, base W is A or T, and base H is A or T or C.

2. The method according to claim 1, characterized in that: The reaction system for PCR amplification included: 100 ng of environmental DNA sample, 5.5 μL of premixed primers, 1 μL of DNA polymerase, 25 μL of Buffer Mix, and sterile enzyme-free water to 50 μL; Wherein, the premixed primers include 2.5 μL of Index_Ind1-24_COX1 primer pair, 0.5 μL of Index_Ind1-24_16S primer pair, and 2.5 μL of Index_Ind1-24_CYTB primer pair; The reaction conditions of the PCR amplification are: 94°C for 1 min; 98°C for 10 s, 45°C for 15 s, 68°C for 15 s, 20 cycles; 98°C for 10 s, 50°C for 15 s, 68°C for 15 s, 15 cycles; 68°C for 5 min, and stored at 12°C.

3. The method according to claim 2, characterized in that The labeling strategy for double-end sample labeling includes the following steps: Using the primers optimized for the detection target and with sample labels, the PCR amplification reaction system and PCR amplification reaction conditions described in claim 2, and the environmental DNA sample from seawater as a template, single-tube one-step PCR amplification is performed.

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