AU2021100290A4 - A new method for monitoring Fenneropenaeus chinensis - Google Patents
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Abstract
The invention provides a rapid and accurate investigation method for the spatio-temporal
distribution and population dynamic change of released Fenneropenaeus chinensis, and the
detection method for released individuals is completed by specifically amplifying DNA
released by Fenneropenaeus chinensis in water environment, which mainly comes from
excrement of Fenneropenaeus chinensis and shedding of shrimp bodies, and can play an
effective detection effect on Fenneropenaeus chinensis in any growth period without collecting
Fenneropenaeus chinensis samples. Real-time fluorescence quantitative PCR can not only
judge the existence of Fenneropenaeus chinensis, but also infer the biomass of
Fenneropenaeus chinensis from the copy number of DNA released by Fenneropenaeus
chinensis. It can quickly and accurately detect the temporal and spatial distribution and
population dynamics ofFenneropenaeus chinensis, and then evaluate the effect of proliferation
and release.
Description
A new method for monitoring Fenneropenaeus chinensis
[01] The invention belongs to the technical field of releasing marine animals, in particular to a new method for monitoring Fenneropenaeuschinensis, that is, a new method for accurately investigating the temporal and spatial distribution and resource dynamic changes of releasing Fenneropenaeuschinensis.
[02] Fenneropenaeuschinensis is a unique annual large-scale economic shrimp in China, which is mainly distributed in the coastal waters of the Yellow Sea and Bohai Sea, which has remarkable economic benefits. Since 1980s, due to the comprehensive factors such as increasing fishing intensity, ecological changes, water pollution, frequent diseases and the blind demand of artificial breeding for wild Fenneropenaeus chinensis, the wild resources of Fenneropenaeus chinensis have shrunk sharply. In order to protect and restore the population and fishery resources of Fenneropenaeus chinensis, China began to migrate/multiply and release Fenneropenaeus chinensis in specific sea areas in 1984, releasing about 3 billion larvae every year.
[03] After many years of large-scale seedling release, the resources of Fenneropenaeus chinensis have been restored to some extent. However, due to the inability to accurately grasp the spatial and temporal distribution and dynamic changes of the released population, it is impossible to scientifically evaluate the supplement level of released population to wild resources, analyze the dynamic changes of wild resources and formulate a scientific release plan, which is an urgent problem to be solved in the release of Fenneropenaeuschinensis. Accurately grasping the temporal and spatial distribution of species and the dynamic changes of population is the prerequisite for rational utilization and development of fishery resources. However, for researchers and policy makers, it is the biggest challenge to collect accurate data on population distribution and population dynamics of aquatic organisms. Traditional resource investigation methods are time-consuming and laborious, and have low sensitivity, which often cannot truly reflect the temporal and spatial distribution of Fenneropenaeuschinensis and the dynamic changes of population resources. This is mainly due to the life history types of Fenneropenaeuschinensis: 1) In the early stage of proliferation and release, the background resources of Fenneropenaeuschinensis are scarce, and the traditional resource investigation methods are inefficient and ineffective; 2) In the early stage of proliferation and release, the swimming ability of Fenneropenaeus chinensis was weak, and the information about its population distribution and dynamic changes of resources could not be accurately obtained by traditional investigation methods; 3) At present, the investigation of fishery resources in China is mainly based on bottom trawl, but bottom trawl is not the investigation method of fishery resources of Fenneropenaeus chinensis. Therefore, it is very important to explore a method that can accurately detect the temporal and spatial distribution and dynamic changes of resources of Fenneropenaeuschinensis. And the method should have the following characteristics: 1) It can accurately detect Fenneropenaeuschinensis without interference from other species; 2) The final result of detection is not affected by subjective factors.
[04] The technical problem to be solved by the invention is to provide a rapid and accurate investigation method for the temporal and spatial distribution and population dynamic changes of released Fenneropenaeuschinensis, so as to master the distribution and population dynamic changes of released Fenneropenaeuschinensis in natural sea areas, accurately evaluate the release effect of Fenneropenaeuschinensis, and further formulate a scientific release plan and effectively promote the recovery of wild Fenneropenaeuschinensis population resources.
[05] The method for investigating released Fenneropenaeuschinensis resources of the invention comprises the following steps:
[06] 1) After Fenneropenaeus chinensis was released, water samples were collected in the investigated sea area, and 2L of seawater was filtered by 0.45tm glass fiber membrane combined with vacuum filtration device for sample enrichment. The collected samples were prepared for Edna extraction;
[07] 2) The Edna of the sample collected in step 1) was extracted and diluted to the concentration range required by quantitative PCR;
[08] 3) The standard curve and amplification curve of mtDNA COI gene of Fenneropenaeus chinensis were generated by fluorescence quantitative detection system. The copy number of mtDNA COI gene of Fenneropenaeuschinensis in the samples with unknown Edna concentration was automatically obtained, and the temporal and spatial distribution and resource dynamics of Fenneropenaeuschinensis in the natural sea area were finally determined according to the results of quantitative PCR.
[09] The sequence of primers and probes of real-time fluorescent quantitative PCR in step 3) is as follows:
[010] The forward sequence COI DF: AGGGGGTAGGAACAGGAAC (SEQ ID NO: 1) ;
[011] Reverse sequence COI DR: GACACAGCTAGATGCAGCG (SEQ ID NO: 2) ;
[012] The probe: 5 ' FAM-TCAGTATATTGCTCATGCCGAGCTTCAGT-3 BHQ 1 (SEQ ID NO: 3).
[013] Further, the reaction system of the real-time fluorescent quantitative PCR is 20 L reaction system, including 10 L 2 x TaqMan fast qPCR master Mix, 0.4[tL forward primer of claim 3, 0.4tL reverse primer of claim 3, 0.4tL probe of claim 3, 24L template DNA, 6.8 L PCR grade water.
[014] Further, the amplification reaction procedure of real-time fluorescent quantitative PCR described above is a two-step process: pre denaturation at 94 °C for 3 min; Denaturation at 94 °C for 5s, annealing at 60 °C for 34 s, with 40 cycles.
[015] In the quantitative analysis, the common PCR primers used in the preparation of standard plasmid DNA using the EDNA detection technique of Fenneropenaeuschinensis are as follows :
[016] The forward sequence COI PF is: TtgTagTTACAGCCCACGCT (Seq ID NO: 4),
[017] The reverse sequence COI PR is: AAATTCCGAAGGCGGGT (SEQ ID NO: 5). Furthermore, the reaction system of common PCR in the construction of mtDNA COI gene standard curve of Fenneropenaeus chinensis is: 50 L reaction system, in which 10 x Taq buffer 5 L, 2.5 mm dNTPs 1 L, 10 mm forward and reverse primers 1 L each, 5U /L Taq DNA polymerase 1 L, 50ng /4L template DNA 2L, mm MgCl 23 L, ddH20 36 L.
[018] Additionally, the amplification reaction procedure of common PCR in the construction of mtDNA COI gene standard curve of Fenneropenaeus chinensis is as follows: pre denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 1 min, 35 cycles; re-extension at 72 °C for 10 min; preservation at 4 °C and end of the procedure.
[019] Beneficial effects:
[020] According to the method, accurate detection of all releasing groups in the whole sea area can be realized, and a foundation is laid for the evaluation of the proliferation releasing effect. Compared with the prior art, the advantages are as follows:
[021] High sensitivity, which is not affected by population density and life history characteristics, and has a high detection rate;
[022] It is economical, efficient, time-saving and labor-saving. Compared with traditional methods, this technology requires less material resources, financial resources, manpower and time;
[023] The interference to the ecosystem is low. The technology only needs to analyze the collected water samples to get the results, and the interference to the biology and ecosystem is low;
[024] The requirements for investigators are low. This technology only needs molecular biology methods, but does not require investigators to have strong morphological classification and identification capabilities;
[025] 5) The sampling limitation is small, and the sampling method required by this technology is simple and low, and the external influence factors are less.
[026] The method for detecting released individuals of the invention is completed by specifically amplifying DNA released by Fenneropenaeus chinensis in water environment, which mainly comes from excrement of Fenneropenaeuschinensis and shedding of shrimp body, and can effectively detect Fenneropenaeuschinensis in any growth period, and does not need to collect any Fenneropenaeus chinensis samples, therefore, not only can the existence of Fenneropenaeuschinensis be judged by real time fluorescence quantitative PCR, Moreover, the biomass of Fenneropenaeus chinensis can be inferred from the copy number of DNA released by Fenneropenaeus chinensis, and the temporal and spatial distribution and population dynamic changes of Fenneropenaeuschinensis can be detected quickly and accurately.
[027] Fig. 1: The relationship between eDNA degradation and time of Fenneropenaeuschinensis;
[028] Fig. 2: Concentration diagram of eDNA enriched by different filter membrane types and filtered water volume;
[029] Fig. 3: The output diagram of eDNA enriched by different filter membrane types and filtered water volume;
[030] Fig. 4: Investigation of Fenneropenaeuschinensis in Bohai Sea in June, 2017 by the method of the present invention.
[031] In order to solve the problems in the background technology, there are two conditions: 1) it can accurately detect Fenneropenaeuschinensis without interference from other related species of Penaeus; 2) The final result of detection will not be affected by subjective factors, and the misjudgment of results will not be caused by human factors. EDNA(environmental DNA,eDNA) refers to free DNA molecules released from skin, mucus, saliva, sperm, secretions, eggs, feces, urine, blood, roots, leaves, fruits, pollen and decayed bodies. According to the invention, the eDNA of collected samples is detected, the eDNA of released Fenneropenaeus chinensis is effectively collected by an established method, the eDNA from the marine environment is amplified by real-time fluorescence quantitative PCR(TaqMan probe method) through a designed highly specific primer, and relevant qualitative and quantitative analysis research is carried out according to the final result of PCR reaction, so that the temporal and spatial distribution and the dynamic change of the population of Fenneropenaeuschinensis in the natural sea area can be inferred.
[032] The following describes the present invention in detail with examples.
[033] Example 1: Establishment and optimization of detection technology for eDNA of Fenneropenaeuschinensis
[034] Detection of retention time of Fenneropenaeuschinensis eDNA in water
[035] EDNA is extremely easy to degrade and has very low content in the environment, so its retention time in the environment will directly affect the later qualitative and quantitative analysis. In order to be able to accurately apply the eDNA technology to the research field of aquatic ecosystems, it is particularly important to explore the retention time of eDNA in water. Generally speaking, the retention time of eDNA released by aquatic animals in water is 7-30 days. However, different biological species have different life history characteristics, different release rates and different amounts of eDNA and sizes of eDNA fragments. Therefore, for different species, their released DNA has different retention time in the environment. The applicant's research found that, taking Fenneropenaeuschinensis as the research object, 25L of water samples were taken from the Fenneropenaeuschinensis culture pond and brought back to the laboratory with sterile white plastic barrels sterilized in advance. The water samples were stored at room temperature (the barrels were kept inflated with an air pump until the sampling was completed). During this period, 15ml of water samples were taken from the barrels every 24 hours and put into 50ml sterile centrifuge tubes. At the same time, 3mol/L of sodium acetate solution 1.5ml and anhydrous ethanol 33ml were added into the centrifuge tubes. Three parallel samples were taken at each time and stored at -20°C until the extraction of eDNA. after the extraction of eDNA, real time fluorescence quantitative PCR(TaqMan probe method) was applied to quantitatively analyze the degradation of eDNA in water environment over time. the results showed that when the release source of eDNA was removed, the content of eDNA in water had a negative correlation with time, and its retention time in the environment was about one month (Fig. 1). By exploring the retention time of DNA released into water by Fenneropenaeuschinensis, the qualitative% a of species can be reasonably planned in the later stage.
[036] 2. Establishment and Optimization of the Operation Process of eDNA Technology
[037] In the detection of eDNA, the amount of eDNA released varies with the size of the eDNA fragment due to the differences between species. Therefore, that invention design different methods for enrich and extracting the eDNA, so as to achieve the best research effect. Taking Fenneropenaeus chinensis as the research object, the following scheme was designed: enrichment of eDNA by filtration membrane method, selection of glass fiber membrane, nitric acid fiber membrane, polycarbonate membrane and nylon membrane with a diameter of 47mm, each of which was set with 4 gradients of 0.45[tm, 0.8[tm, 1.2tm and 5[m according to its pore size, sampling water volume was set with 3 gradients of 500mL, IL and 2L, and eDNA was extracted by dn easy blood and tissuekit (qiagen, hilden, Germany) kit. After the extraction of the eDNA is completed, the copy number of the enriched eDNA on each filter membrane is detected by real-time fluorescence quantitative PCR(TaqMan probe method), and the type of filter membrane, the pore size of the filter membrane and the volume of water sample which are most suitable for the enrichment of the eDNA of Fenneropenaeus chinensis are determined according to the size of the copy number. The final results show that the material of the filter membrane, the pore size of the filter membrane and the volume of the water sample have certain influence on the qualitative and quantitative analysis ofFenneropenaeuschinensis, among which the 0.45 m glass fiber filter membrane can detect the highest concentration of DNA in 2L water sample (Fig. 2), which is 1750copies/[tL, and its eDNA yield is also the highest (fig. 3). based on this, a set of operation process of Fenneropenaeus chinensis eDNA technology is established, which improves the detection rate of Fenneropenaeus chinensis and provides a basis for the subsequent distribution monitoring and biomass assessment of Fenneropenaeuschinensis.
[038] Example 2: Application of Edna detection technology of Fenneropenaeus chinensis in Bohai ecosystem
[039] In June, 2017, during the investigation of fishery resources in Bohai Sea, the temporal and spatial distribution of Fenneropenaeuschinensis released in Bohai Sea was detected and the relative resources were analyzed by using the method of the present invention. The specific implementation plan is as below.
[040] According to the 54 investigation stations set up during the fishery resources investigation, 2L surface seawater (Om-1.0m) is taken from each station, filtered by a 0.45tm glass fiber filter membrane combined with a vacuum filtration device, and eDNA enrichment is carried out; each station is technically repeated for three times; after filtration, each filter membrane sample is rolled up and wrapped in an independent foil paper, and stored in the dark at -20°C and brought back to the laboratory. EDNA was extracted by DneasyBlood and Tissue Kit (Qiagen, Hilden, Germany) in laboratory, and diluted to the concentration range required by real-time fluorescence quantitative PCR. The genomic DNA of Fenneropenaeuschinensis was extracted by the traditional phenol-chloroform-isoamyl alcohol method. after extraction, the genomic DNA was amplified by PCR with primer pair COI PF/COI PR (see table 1 for sequence) (the reaction system of PCR was 50[1, including 10xTaq Buffer 5 L, dNTPs (2.5mM)1 L each) I 1, 1 L for forward and reverse primers (COI PF/COI PR)(10mM), 1 1 for Taq DNA polymerase (5u/j 1), 2 1 for template DNA (50ng/j 1), 3 1 for MgCl2 (25mm) and 36 1 for ddH20; The amplification reaction procedure of PCR: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 1min, 35 cycles; re-extension at 72°C for 10min; Save the samples at 4°C and end the program. Purify the PCR product, connect it to pMD-18-T plasmid vector, transform it into competent cells of Escherichia coli to construct pMD-18-T-COI recombinant plasmid, culture it overnight on LB solid plate culture medium, select single colony for expanded culture to extract recombinant plasmid DNA, and finally dilute the recombinant plasmid DNA to a specific concentration as standard DNA. Then, the quantitative analysis of eDNA was carried out by the 2x TaqMan fast qpcr master mix (lowrox) real-time fluorescence quantitative PCR kit of BBI Life Sciences Co., Ltd. combined with the primer pair COI DF/COI DR (sequence in Table 1) (the reaction system of PCR is 201, Including 10tL 2xTaqMan Fast qPCR Master Mix, 0.4tL Forward primer (10 mol L-1), 0.4[tL Reverse primer (10 mol sequence), 0.4tL probe (10 mol sequence), 2tL template DNA, 6.8 1 PCR-grade water; The amplification reaction procedure of PCR is two steps: pre-denaturation at 94°C for 3 min; Denaturing at 94°C for 5s, annealing at 60°C for 34s, 40 cycles. ), The final result shows that all the 54 stations set up in the Bohai Sea fishery resources survey can detect Fenneropenaeuschinensis (Figure 4), while the bottom trawl survey results show that only one station can detect Fenneropenaeus chinensis and only one Fenneropenaeuschinensis (not released in that year), which proves that this method has great potential in fishery resources survey.
[041] Table 1 Primer information of mtDNA COI gene PCR amplification in Fenneropenaeuschinensis
Anneling Fragment Prmer Primer sequence (5- 3) temperatur Ae(P
CO] PR AAAT ATiOAMk G 7 C01]DF AGGGGT AA(AUAGUA AC 7 10
COI DR GACACCAGCTLAGAC 5. Prob~ 5' FAM-TCAGFTAGAATTGTATYGAGCTTCA GT-3' RlQl 6.2 106
[042] The results show that the method of the present invention has great potential in the detection of Fenneropenaeus chinensis, which is very suitable for the investigation of fishery resources. It has extremely high sensitivity that bottom trawl does not have, and has a very wide application prospect in the detection of temporal and spatial distribution and population dynamic change of Fenneropenaeuschinensis. It can be applied to the spatio-temporal distribution detection and population dynamic change detection of released individuals in the releasing practice of Fenneropenaeus chinensis, in order to realize the accurate evaluation of releasing effect.
[043] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, in keeping with the broad principles and the spirit of the invention described herein.
[044] The present invention and the described embodiments specifically include the best method known to the applicant of performing the invention. The present invention and the described preferred embodiments specifically include at least one feature that is industrially applicable
Claims (9)
1. A new method for monitoring Fenneropenaeus chinensis, which is characterized in that the method comprises the following steps
1) After Fenneropenaeus chinensis was released, water samples were collected in the investigated sea area, and 2L of seawater was filtered by 0.45tm glass fiber membrane combined with vacuum filtration device for sample enrichment. The collected samples were prepared for Edna extraction;
2) The Edna of the sample collected in step 1) was extracted and diluted to the concentration range required by quantitative PCR;
3) The standard curve and amplification curve of mtDNA COI gene of Fenneropenaeus chinensis were generated by fluorescence quantitative detection system. The copy number of mtDNA COI gene of Fenneropenaeus chinensis in the samples with unknown Edna concentration was automatically obtained, and the temporal and spatial distribution and resource dynamics of Fenneropenaeuschinensis in the natural sea area were finally determined according to the results of quantitative PCR.
2. The method according to claim 1, which is characterized in that step 1) in which each sampling point is sampled three times repeatedly.
3. The method according to claim 1, which is characterized in that the sequence of primers and probes of real-time fluorescent quantitative PCR in step 3) is as follows:
The sequence of the forward primer was SEQ ID No: 1, the sequence of the reverse primer was SEQ ID No: 2, and the sequence of the probe was SEQ ID No: 3.
4. The method according to claim 3, which is characterized in that the 5 'end of the probe is marked with FAM and the 3' end is marked with BHQ11.
5. The method according to claim 1, which is characterized in that in step 3), the reaction system of the real-time fluorescent quantitative PCR is 20L reaction system, including 10jL 2 x TaqMan fast qPCR master Mix, 0.4tL forward primer of claim 3, 0.4tL reverse primer of claim 3, 0.4tL probe of claim 3, 2tL template DNA, 6.8[tL
PCR grade water.
6. The method according to claim 1, which is characterized in that the amplification reaction procedure of real-time fluorescent quantitative PCR described in step 3) is a two-step process: pre denaturation at 94 °C for 3 min; Denaturation at 94 °C for 5s, annealing at 60 °C for 34 s, with 40 cycles.
7. The method according to claim 1, which is characterized in that in the step 3), the common PCR primers used in the construction of the mtDNA COI gene standard curve of Fenneropenaeuschinensis, wherein the sequence of the forward primer is SEQ ID No: 4, and the sequence of the reverse primer is SEQ ID NO: 5.
8. The method according to claim 1, which is characterized in that in step 3), the reaction system of common PCR in the construction of mtDNA COI gene standard curve of Fenneropenaeuschinensis is: 50 L reaction system, in which 10 x Taq buffer pL, 2.5 mm dNTPs 1 L, 10 mm forward and reverse primers 1 L each, 5U /4L Taq DNA polymerase 1 L, 5Ong /4L template DNA 2L, 25 mm MgCl 23 L, ddH20 36 L.
9. The method according to claim 1, which is characterized in that in step 3), the amplification reaction procedure of common PCR in the construction of mtDNA COI gene standard curve of Fenneropenaeuschinensis is as follows: pre denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 1 min, 35 cycles; re-extension at 72 °C for 10 min; preservation at 4 °C and end of the procedure.
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