CN113981107A

CN113981107A - Primer combination for detecting environment DNA of yellow fin spine porgy and application thereof

Info

Publication number: CN113981107A
Application number: CN202111353738.2A
Authority: CN
Inventors: 秦传新; 孙美婧; 郭禹
Original assignee: South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
Current assignee: South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
Priority date: 2021-11-16
Filing date: 2021-11-16
Publication date: 2022-01-28

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a primer combination for detecting environment DNA of oplegnathus fasciatus and application thereof, aiming at the fact that research on eDNA of reefs fishes is less at present, the invention designs a fluorescent primer and a Taqman probe which can verify the target species oplegnathus fasciatus by collecting water samples of living water areas of the oplegnathus fasciatus on the premise of not damaging ecological environment and detecting by using a molecular technology, finally determines a primer combination (a specific primer pair and the probe) of COI genes with strong specificity and high sensitivity, and establishes the concentration of the target species and C genes by absolute quantification_tThe primer combination can be used for identifying whether a target species of the yellow spine porgy exists in a water sample or not, and determining the concentration of the yellow spine porgy in the water sample, and the concentration of the yellow spine porgy in a passing loopThe environmental DNA detection has very important significance in resource protection of the yellow fin spine porgy.

Description

Primer combination for detecting environment DNA of yellow fin spine porgy and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a primer combination for detecting environment DNA of a yellow fin sea bream and application thereof.

Background

The yellow-fin spine sea bream (Acanthopagrus latus) belongs to Pisces of the class Dermatophagoides, Perciformes of the order Perciformes, Sparidae of the family Paciformae, Acanthopagrus of the genus Acanthopagrus, and is widely distributed in the red sea, Arabic, Indian ocean, Western pacific coast, Taiwan, Fujian, Guangdong, and Guangxi coast.

The yellow fin spine sea bream lives in the coastal sea area and the estuary, has omnivorous nature, fresh and tender meat quality and rich nutrition, and is an important economic fish. As a rocky fish, the yellow fin acanthopagrus does not have long-distance migration habit, but has obvious reproductive migration behavior. The culture cycle of the oplegnathus fasciatus is long, the market specification of about 5 can be reached only after one year, a half year or two years, and the enthusiasm of fishermen for culturing the oplegnathus fasciatus is greatly limited. In recent years, the natural population of the yellow fin spine porgy is seriously degraded, the germplasm is seriously degraded and the like due to environmental pollution, over-fishing and the like. Therefore, the resource protection of the yellow fin spine porgy has important significance. The distribution pattern and the population size of the yellow fin spine porgy population are mastered in time, and the method is important for resource protection and biodiversity research of the yellow fin spine porgy.

For some populations with extremely low density and special life history, it is very difficult to detect the space-time distribution and investigate the population dynamic change. The environmental DNA (eDNA) technology is a method for qualitative or quantitative analysis by using various molecular biological techniques after directly extracting a target gene fragment from an environmental sample (such as soil, sediment, water, etc.). This method was first in the 80's of the last century. In the next 20 years, with the breakthrough of key technologies such as sample sampling, DNA extraction, analysis and sequencing, the eDNA analysis is developed into a complete species distribution detection technical means. In recent years, the eDNA technology has been widely applied to the research of aquatic organism protection monitoring, including many endangered species, invasive species and other special populations. Fluorescent Quantitative PCR (qPCR) has high specificity, can quantitatively analyze DNA, and has become an important means for eDNA analysis in recent years instead of general PCR.

At present, research on the eDNA of the reef fishes is less, and no report is found about the research on the eDNA specific amplification primer and the fluorescent probe of the oplegnathus fasciatus. Therefore, establishing a detection method of the oplegnathus fasciatus eDNA and realizing simple, quick, accurate and objective molecular identification of the oplegnathus fasciatus are important contents for developing resource protection of the oplegnathus fasciatus at present.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides the primer combination for detecting the environmental DNA of the yellow fin spine porgy, can be used for identifying whether the target species of the yellow fin spine porgy exists in a water sample and determining the concentration of the yellow fin spine porgy in the water sample, and has very important significance for developing the resource protection of the yellow fin spine porgy through the environmental DNA detection.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a primer combination for detecting environment DNA of a yellow fin spine porgy, which comprises a specific primer and a probe, wherein the nucleotide sequence of the specific primer is respectively shown as SEQ ID NO: 9 and SEQ ID NO: 10, and the nucleotide sequences of the probes are respectively shown as SEQ ID NO: shown at 13.

The invention also provides application of the primer combination in detecting the environmental DNA of the yellow fin spine porgy.

The tissue and water sample of the oplegnathus fasciatus are subjected to PCR and agarose gel electrophoresis to verify the specificity and sensitivity of each pair of primers, the probe is designed according to the specific primer, and the sensitivity of the probe is verified through qPCR to finally obtain a pair of specific primers and probes capable of detecting the oplegnathus fasciatus in the water sample.

The invention also provides a kit for detecting the environmental DNA of the yellow fin spine porgy, and the kit comprises the specific primer.

The invention also provides a fluorescent quantitative kit for detecting the environmental DNA of the yellow fin spine porgy, and the kit comprises the primer combination.

The invention also provides a method for detecting the yellow fin spine porgy by the eDNA technology, which comprises the following steps: the specific primer is used for carrying out PCR detection on DNA of the living environment of the yellow fin sea bream, and whether the target species of the yellow fin sea bream exists in the water sample is determined and identified according to the existence of the detection strip.

Preferably, the PCR assay uses a 50 μ L system comprising 2 XTAQQ Mix (1X)25 μ L, DNA template (<500ng)1-5 μ L, Primer F (0.1-1.0uM)1 μ L, Primer R (0.1-1.0uM)1 μ L, sterile water to 50 μ L.

Preferably, the reaction procedure of the PCR detection adopts a 3-step method, and is specifically set as follows: (1) pre-denaturation at 94 ℃ for 3 min; (2) denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and amplification for 35 cycles; (3) further extension was carried out at 72 ℃ for 5 min.

The invention also provides a PCR fluorescent quantitative method suitable for the red sea bream eDNA, which specifically comprises the following steps: and (3) carrying out fluorescent quantitative PCR on DNA of the living environment of the yellow fin sea bream by using the primer combination, drawing a standard curve between the concentration of the standard substance of the CO I gene plasmid of the yellow fin sea bream and the Ct value, and finally determining the concentration of the DNA of the yellow fin sea bream environment according to the standard curve.

Preferably, the reaction system of the fluorescent quantitative PCR adopts a 20-L system, comprising 2 xqPCR Mix 10. mu.L, forward primer (10. mu. mol/L) 0.4. mu.L, reverse primer (10. mu. mol/L) 0.4. mu.L, probe (10. mu. mol/L) 0.4. mu.L, template DNA 2. mu.L and PCR water 6.8. mu.L.

Preferably, the amplification reaction procedure of the fluorescence quantitative PCR adopts a two-step method, and is specifically set as follows: (1) pre-denaturation at 94 ℃ for 3 min; (2) denaturation at 94 ℃ for 5s, annealing and extension at 60 ℃ for 30s, and 40 cycles.

Compared with the prior art, the invention has the beneficial effects that:

aiming at the current few researches on the eDNA of the rocky fish, the invention designs a fluorescent primer and a Taqman probe which can verify the target species of the yellow fin sea bream by collecting a water sample of the living water area of the yellow fin sea bream and detecting by using a molecular technology on the premise of not damaging the ecological environment, finally determines a primer combination (a specific primer pair and the probe) of a COI gene with strong specificity and high sensitivity, and establishes the concentration and C of the target species by absolute quantification_tThe primer combination can be used for identifying whether a target species of the sparus latus exists in a water sample or not, can determine the concentration of the sparus latus in the water sample, and has very important significance for developing resource protection of the sparus latus through environmental DNA detection.

Drawings

FIG. 1 shows the results of agarose gel electrophoresis of PCR products (the first five are DNAs of muscle tissue and the second five are water-sampled eDNAs);

FIG. 2 shows the results of PCR and electrophoresis of the intestinal DNA of yellow fin sea bream at 55 ℃ annealing temperature;

FIG. 3 shows the results of PCR and electrophoresis of intestinal DNA of yellow fin sea bream at an annealing temperature of 58 ℃;

FIG. 4 shows the results of PCR and electrophoresis of the DNA of a water sample at an annealing temperature of 55 ℃;

FIG. 5 shows the results of PCR and electrophoresis of the DNA of a water sample at an annealing temperature of 58 ℃;

FIG. 6 is a qPCR standard curve of the mtDNA COI gene of yellow fin sea bream.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

Example 1 establishment of an environmental DNA detection System for yellow fin sea bream

First, primer design

The sparus latus mitochondrial DNA (mtDNA) COI gene fragment (GenBank accession number: JX983210.1) was searched in GenBank database, 5 pairs of primers were designed using Primer express3.0 and synthesized by Biotechnology (Shanghai) GmbH, and 5 pairs of specific primers and 1 pair of general primers were designed in total, including COI COI P (F: SEQ ID NO: 1, R: SEQ ID NO: 2), COI QF1 (F: SEQ ID NO: 3, R: SEQ ID NO: 4), COI QF2 (F: SEQ ID NO: 5, R: SEQ ID NO: 6), COI QF3 (F: SEQ ID NO: 7, R: SEQ ID NO: 8), COI 4 (F: SEQ ID NO: 9, R: SEQ ID NO: 10), COI QF5 (F: SEQ ID NO: 11, R: SEQ ID NO: 12), and Table 1 shows. Wherein, the universal PCR primer is used for preparing plasmid standard product DNA, and the target segment amplified by the specific primer is a part of the target segment amplified by the common PCR primer.

Primers designed in Table 1

Second, verification of specific primers

1. Extraction of DNA (eDNA)

The extraction was performed using the DNeasy Blood and Tissue Kit (Qiagen, Germany). The preparation work before extraction includes: (1) all centrifugation steps were carried out at room temperature (15-25 ℃); (2) dissolving the precipitate in Buffer AL and Buffer ATL; (3) adding ethanol to Buffer AW1 and Buffer AW 2; (4) bringing the frozen tissue or cell mass to ambient temperature; (5) the water bath kettle is heated to 56 ℃ in advance; (6) all operations were performed on a clean bench (half an hour before the experiment with ultraviolet light sterilization); (7) sterilizing scissors, tweezers and required gun heads in advance and drying.

1.1 extraction of DNA from muscle tissue of yellow fin spine sea bream

(1) Cutting the tissue (muscle, liver, intestine and stomach, less than or equal to 25mg) of yellow fin acanthopagrus latus (collected from Guangdong Yangjiang) into small pieces, and putting into a 2mL centrifuge tube;

(2) adding 180uL of Buffer ATL, adding 20uL of protease K, mixing uniformly by vortex, carrying out water bath at 56 ℃ for 6h until the tissue is completely cracked, slightly reversing the mixing centrifuge tube every 30min during the water bath to accelerate the cracking, and carrying out vortex oscillation for 15s after the cracking is finished;

(3) adding 200uL Buffer AL, mixing uniformly by vortex, and organizing the sample in a 56 ℃ water bath for 10 min;

(4) adding 200uL (concentration is 96-100) of absolute ethyl alcohol, and uniformly mixing by vortex;

(5) transferring the mixture to DNeasy Mini filter column in 2mL centrifuge tube with pipette gun, centrifuging at 6000 Xg (8000rpm) for 1min 30s, and discarding filtrate and centrifuge tube;

(6) placing DNeasy in 1 new collection tube, adding 500uL of Buffer AW1, centrifuging at 6000 Xg (8000rpm) for 1min 30s, and discarding filtrate and centrifuge tube;

(7) DNeasy was placed in 1 new collection tube, 500uL of Buffer AW2 was added, the tube was centrifuged at 20000 Xg (14000rpm) for 3min 45s, and the filtrate and centrifuge tube were discarded;

(8) placing DNeasy MINI spin column in a new 2mL centrifuge tube;

(9) add 100uL of Buffer AE to DNeasy MiNi spin column to elute DNA, let stand at room temperature (15-25 ℃) for 1min, centrifuge at ≥ 6000 Xg (8000rpm) for l min 30 s.

Immediately after the DNA extraction, the concentration and quality of the sample are checked by using an ultra-micro ultraviolet spectrophotometer, and the eDNA sample is diluted if the concentration of the eDNA sample is higher than 50 ng/. mu.L. mu.L of DNA solution was pipetted into each DNA sample for agarose gel electrophoresis detection and PCR quantification, and the remaining 195. mu.L of DNA solution was stored at-80 ℃ for further use.

1.2 aqueous eDNA extraction

The invention verifies whether the red sea bream is the red sea bream or not by detecting the DNA of a water sample of a target species, so that the red sea bream needs to be collected for detection (the water sample is collected from a water bucket in a central workshop of a tropical aquatic product center of Hainan Ling water, and the red sea bream is cultured in the water bucket).

(1) Selecting a whatman nitrocellulose filter membrane with the diameter of 47mm and the pore diameter of 0.45 mu m to filter a water sample containing a target species, taking the filter membrane out of a refrigerator, removing redundant parts at the edge of the filter membrane by using scissors, cutting the filter membrane into strips, placing the strips into a 2mL sterile centrifuge tube, adding 570 mu L of Buffer ATL and 60 mu L of protease K solution (20mg/mL) into the centrifuge tube, carrying out vortex oscillation, uniformly mixing the mixed solution, carrying out 56 ℃ water bath in a constant-temperature water bath kettle for 6 hours, slightly reversing the centrifuge tube and uniformly mixing the mixed solution every 15min during the water bath period to ensure that the centrifuge tube is fully cracked, taking out the filter membrane after the cracking is finished, and squeezing the rest filter membrane;

(2) adding 630 mu L of Buffer AL into the centrifugal tube by using a liquid transfer gun, and carrying out vortex oscillation for 15s to uniformly mix the Buffer AL and the centrifugal tube;

(3) adding 630 mu L of absolute ethyl alcohol into the centrifugal tube by using a liquid transfer gun, and carrying out vortex oscillation for 15s to uniformly mix the absolute ethyl alcohol and the centrifugal tube;

(4) transferring the mixed solution in a 2mL centrifuge tube into a DNeasy centrifugal column for 3 times, and centrifuging for 1min and 30s at 25 ℃ and 8000 g;

(5) placing the column into a new 2mL collection tube, adding 500. mu.L Buffer AW1 (treated as per the instructions) into the column with a pipette, and centrifuging at 25 deg.C and 8000g for 1 min;

(6) placing the column into a new 2mL collection tube, adding 500 μ L Buffer AW2 (treated according to the instruction) into the column with a pipette, and centrifuging at 25 deg.C and 8000g for 3min 45 s;

(7) placing the centrifugal column into a 1.5mL sterile centrifuge tube, adding 100 μ L Buffer AE in the center of the centrifugal column, incubating at 25 deg.C for 1mi, and centrifuging at 25 deg.C and 8000g for 1min 30 s;

immediately after the extraction of the eDNA, an ultramicro ultraviolet spectrophotometer is used for detecting the concentration and the quality of the eDNA, and if the concentration of the eDNA sample is higher than 250 ng/muL (the real-time fluorescence quantitative PCR kit requires that the concentration of the DNA of the sample to be detected is lower than 250 ng/muL), the eDNA sample is diluted. mu.L of DNA solution was pipetted into each eDNA sample for detection by agarose gel electrophoresis and quantitative PCR analysis, and the remaining 195. mu.L of eDNA solution was stored at-80 ℃ for further use.

2. PCR reaction system and agarose gel electrophoresis

2.1PCR amplification

PCR amplification was performed using 2 XTaq PCR MasterMix II kit using a 50. mu.L system including 2 XTaq MasterMix (1X) 25. mu.L, DNA template: (<500ng)1-5 μ L, Primer F (0.1-1.0uM)1 μ L, Primer R (0.1-1.0uM)1 μ L, adding sterile water to 50 μ L. The reaction procedure adopts a 3-step method, and is specifically set as follows: (1) pre-denaturation at 94 ℃ for 3 min; (2) denaturation at 94 ℃ for 30s, annealing at 55 ℃ (subsequent experimental verification is needed) for 30s, extension at 72 ℃ for 1min, and amplification for 35 cycles; (3) further extension was carried out at 72 ℃ for 5 min. And increased by ddH₂O is a negative control for the template.

Mixing the mixed solution with the total volume of 50 mu L in a PCR tube according to the ratio of the reagents (the specificity of different primers needs to be verified in the next step of electrophoresis, so that different primers are added for PCR amplification), performing the preparation process on ice (4 ℃), mixing the mixed solution after the preparation is finished, putting the mixed solution into a T960 PCR instrument for PCR, and taking out a reaction product for agarose gel electrophoresis after the preparation is finished.

2.2 agarose gel electrophoresis

5 μ L of PCR amplified product was detected by agarose gel electrophoresis.

Preparing glue according to the molecular weight, adding SYBR Safe DNA Gel Stain, adding DL2,000DNA Marker in the first hole, adding 6 × loading buffer in the sample, mixing, spotting, connecting the positive electrode and the negative electrode, setting voltage, and running glue (clean glue preparation plate and electrophoresis tank):

(1) weighing: 0.3g agarose +30ml TAE buffer, placed in a triangular flask;

(2) melting glue: mixing the weighed agar powder and TAE in a conical flask, heating the mixture for one half in a microwave oven until the agar powder is dissolved and has no bubbles (uniformly shaking once in about 30 s);

(3) pouring glue: placing a comb in a clean glue groove, dripping about 3uL of nucleic acid dye (ethidium bromide is added) (no bubbles exist) into the glue, mixing uniformly, waiting for 30min, cooling to be not hot, and pouring the mixture into a glue plate lightly;

(4) pulling out a comb: after the comb is pulled out for about 20 minutes, if the comb is not pulled out well, a proper amount of TAE buffer solution can be dripped;

(5) sample application: spotting 5uL DNA (PCR amplified product) in sample wells, DL2,000DNA Marker 3uL (one well), starting in black, negative side;

(6) glue running: setting U as 130V, V as 150mA, and I as 30 min;

(7) the Gel was placed into a Gel imaging system (300nm) and the computer turned on Gel-Pro-Analyzer → image acquisition → scanning.

The PCR product is detected by agarose gel electrophoresis, the result is shown in figure 1, the designed universal primer and the designed specific primer pair can successfully amplify the designed target fragment, the expected result is proved to be completely consistent, the electrophoresis band is single and bright, no miscellaneous band is shown, and the designed primer has good specificity.

3. Specificity and sensitivity of primers are verified through double verification of red-fin sea bream tissues and water samples

(1) Tissue of

Firstly, DNA of 6-tailed yellow fin sea bream tissues (muscle, liver, intestine and stomach) is extracted, the concentration is shown in table 2, and the intestine with better purity and concentration is selected for subsequent experimental verification.

TABLE 2 purity and concentration of DNA from different tissues of yellow fin spine sea bream

Tissue of	Detection of purity (A)₂₆₀/₂₈₀)	Concentration (ng/. mu.L)
			Muscle	1.83	16.33
Liver disease	1.76	314.22
			Sausage	2.00	250.25
Stomach (stomach)	2.10	173.13

The purity of DNA is generally better at 1.8-2.0, comparing the purity A of four tissues₂₆₀/₂₈₀And concentration, the intestine was selected for subsequent experiments. By carrying out gel running on the PCR reaction product of the intestinal DNA of the yellow fin sea bream, no strip is run out of QF1, QF2 and QF3, and the three pairs of primers are excluded. QF4 and QF5 could run relatively clear bands, but the QF5 primer had dimers (negative control results)The presence of banding) is excluded.

Specifically, the annealing temperature was set to 55 ℃, QF4 and QF5 could run out of bands (the reaction system, procedure and amplification results are shown in fig. 2), PCR was performed according to the instructions of 2 × Taq PCR MasterMix ii kit (containing dyes), the primer annealing temperature was set to 58 ℃, both pairs of primers QF4 and QF5 could run out of bands, and the bands were clearer (the reaction system, procedure and amplification results are shown in fig. 3).

(2) Water sample

By collecting a water sample of the yellow fin spine porgy (in 150L white barrels of a central workshop for aquatic products in the Hannan Ling water and tropical, 5 yellow fin spine porgy are bred in each barrel, each group is repeated three times, and four groups of different filtered water amounts are collected in each barrel), and extracting DNA, detailed information is shown in table 3, and glue is run after PCR.

TABLE 3 purity and concentration of water sample DNA

Water intake (mL)	Detection of purity (A)₂₆₀/₂₈₀)	Concentration (ng/. mu.L)
			500	2.11	27.26
1000	2.09	47.32
			2000	2.10	82.31
3000	2.07	114.33

The annealing temperature was set at 55 ℃ and both pairs of QF4 and QF5 primers could run out of the band, and the QF5 primer had dimers and was excluded (see FIG. 4 for reaction system, procedure and amplification results).

The annealing temperature was set at 58 ℃ and both pairs of QF4 and QF5 primers could run out of the band, and the QF5 primer had dimers and was excluded (see FIG. 5 for reaction system, procedure and amplification results).

It can be seen from the above analysis that agarose gel electrophoresis is performed on the PCR products of tissues and water samples to verify the specificity of 5 pairs of (QF1, QF2, QF3, QF4, QF5) primers, and the specific primer pair can successfully amplify the designed target fragment, which proves that the specificity of the designed primers is very good, the electrophoresis band is single and bright, and no miscellaneous band is present. The three pairs of the QF1, QF2 and QF3 do not generate a band, so that the two pairs of the QF4 and QF5 are excluded, sterile pure water is added in the glue running process to serve as a negative control, the pair of the QF5 is found to have a dimer, so the primer is excluded, and finally the QF4 primer is determined to have a clear band, is a specific primer capable of identifying the COI gene of the oplegnathus fasciatus, and has the optimal annealing temperature of 55 ℃.

Fourth, PCR fluorescent quantitation

(1) Mixing Taqman probe marked with fluorescein with template DNA, finishing high-temp. denaturation, low-temp. renaturation and thermal circulation of proper-temp. extension, following polymerase chain reaction rule, cutting off Taqman probe complementary-matched with template DNA, dissociating fluorescein in reaction system, under the action of specific light excitation emitting fluorescence, along with the increase of circulation number the amplified target gene fragment is exponentially increased, and real-time detecting its correspondent fluorescence signal intensity changed along with amplification to obtain C_tAnd (4) simultaneously using a plurality of standard products with known template concentration as comparison to obtain the copy number of the target gene of the sample to be detected.

C_tValue (Cycle thres)hold, cycle threshold) means: the number of cycles that the fluorescence signal in each reaction tube undergoes when it reaches a set threshold.

Setting of fluorescence threshold

The fluorescence signal of the first 15 cycles of the PCR reaction was taken as the fluorescence background signal, and the default (default) setting of the fluorescence threshold was 10 times the standard deviation of the fluorescence signal of 3-15 cycles, i.e.: threshold is 10 × SD_cycle3-15。

(2)C_tValue to starting template relationship

The Ct value for each template is linear with the logarithm of the starting copy number of the template, and the formula is as follows:

Ct＝-1/lg^(1+Ex)*lg^X0+lg^N/lg(1+Ex)；

n is the number of cycles of the amplification reaction, X0 is the initial template amount, Ex is the amplification efficiency, and N is the amount of amplification product when the fluorescent amplification signal reaches a threshold intensity.

The greater the initial copy number, C_tThe smaller the value. A standard curve can be constructed using a standard with a known starting copy number, where the abscissa represents the logarithm of the starting copy number and the ordinate represents the C_tThe value is obtained. Thus, as long as C of unknown sample is obtained_tThe initial copy number of the sample can be calculated from the standard curve.

4.1 fluorescent quantitative PCR

All the extracted eDNA samples were quantitatively analyzed using 2 × TaqMan Fast qPCR Master Mix (Low Rox) real-time fluorescent quantitative PCR kit from BBI Life sciences, Inc. The PCR reaction system adopts a 20 mu L system, which comprises 10 mu L of 2 xTaqMan Fast qPCR Master Mix, 0.4 mu L of forward primer QF4(10 mu mol/L), 0.4 mu L of reverse primer QF4(10 mu mol/L), 0.4 mu L of probe (10 mu mol/L), 2 mu L of template DNA and 6.8 mu L of PCR water. The amplification reaction program adopts a two-step method, and is specifically set as follows: (1) pre-denaturation at 94 ℃ for 3 min; (2) denaturation at 94 ℃ for 5s, annealing and extension at 60 ℃ for 30s, and 40 cycles.

4.2 preparation of Standard Curve

And (3) sending the fluorescent quantitative PCR product to a biological product company Limited for sequencing, comparing the sequencing result in NCBI-BLAST to obtain a comparison result of 96-100%, determining the comparison result as a target species of the yellow spine porgy, designing a corresponding probe according to the determined specific primer QF4, and synthesizing (the probe is synthesized by the biological product company Limited according to the determined primer QF 4). The specific primers and probes (SEQ ID NO: 13) selected are shown in Table 4:

TABLE 4 MtDNA COI gene PCR amplification primer information of sparus latus

Meanwhile, the plasmid standard substance is extracted from the production and sent back, and the copy number is measured to be 2.9 multiplied by 10⁸copy/uL, a standard curve was prepared from plasmid standards (synthesized by Biotech Co., Ltd. by general primers) in accordance with 2.9X 10⁸、2.9×10⁷、2.9×10⁶、2.9×10⁵、2.9×10⁴、2.9×10³、2.9×10²Standard sample dilutions were made at a concentration of 2.9X 10 copies/. mu.L. The standard and each eDNA sample were repeated 3 times, i.e. 9 times for each sampling type (i.e. water samples of the same volume were filtered with a filter membrane of the same pore size and same material, 3 qPCR repeats x 3 sampling repeats), the final eDNA copy number was averaged, and the experimental data were analyzed using absolute quantitation. qPCR amplification was performed using a qTOWER3 model fluorescent quantitative PCR instrument and 96-well plates (Thermo Fisher), Ct values were calculated and standard and amplification curves were generated.

The preparation process of the standard curve is as follows:

(1) method for dilution of standard in multiple gradient:

1 × stock solution (plasmid standard substance) and 9 × dilution buffer solution (sterilized pure water) to obtain a standard substance i;

1 × standard substance ii +9 × dilution buffer solution to obtain a standard substance ii;

1 × standard substance iv +9 × dilution buffer solution to obtain standard substance iii

1 × standard substance iii +9 × dilution buffer solution to obtain a standard substance iv;

diluting the buffer solution by 1 × standard substance iv +9 × to obtain a standard substance v;

diluting the buffer solution by 1 × standard substance iv +9 × to obtain a standard substance vi;

1 × standard substance iv +9 × dilution buffer solution to obtain a standard substance vii;

1 × Standard iv +9 × Dilute buffer to get the standard viii.

(2) Firstly, a mixed solution is prepared according to a2 XTaqMan Fast qPCR Master Mix (Low Rox) real-time fluorescent quantitative PCR kit, and a 20 mu L reaction system comprises the following components: 10. mu.L of 2 XPCR reaction premix, 0.4. mu.L of forward and reverse primers 0.4. mu. L, QF4 of probe, and ddH of double distilled water₂O6.8. mu.L, plasmid standard 2. mu.L. According to 8 gradients, each of 5 replicate groups, total 40 replicate wells, so to prepare the mixture 40 × 18 μ l (18 μ l of mixture per replicate well, total amount of mixture of 40 replicate wells) 620 μ l, add all above reagents to a sterilized 22mL centrifuge tube, blow and mix them well repeatedly, and put on ice. The second step requires the preparation of 8 gradients of standards on PCR plates, according to 2.9X 10⁸、2.9×10⁷、2.9×10⁶、2.9×10⁵、2.9×10⁴、2.9×10³、2.9×10²Eight gradients (8 sterile PCR tubes on PCR plate) of 2.9X 10 copies/. mu.L were diluted, and after 8 dilutions were completed, the mixture was whipped and placed on ice for use. And finally, taking out the eight-connection pipe, adding 18 mu L of mixed liquor (prepared in the first step) into each compound hole of the eight-connection pipe, respectively adding standard products (prepared in the second step) with different gradients according to the gradients, blowing, uniformly mixing, covering the eight-connection pipe, and placing the eight-connection pipe on an instant centrifuge for short-time centrifugation. Finally, the samples are respectively put into qTOWER³The kit is arranged according to a kit reaction program (mentioned in 4.1 fluorescence quantification), and then the kit is operated to wait for a result.

A standard curve for absolute quantification, the concentration range covering the concentration interval of the sample: 8 concentration gradients, 5 replicates, outliers were screened.

The relation curve between the concentration logarithm of the standard substance and the Ct value is fitted through the obtained 8 points, as shown in the standard curve of FIG. 6, the research shows that the standard substance has a good linear relation in the concentration range of the diluted plasmid standard substance, and the established standard curve can correctly reflect the yellow fin thornAmplification of Pagrus CO I gene. Samples can be prepared by using qTOWER³C obtained by fluorescent quantitative PCR instrument_tAnd obtaining the copy number of the sample through a formula of a standard curve.

The formula for calculating the copy number of the standard plasmid is:

copies ═ 6.02 × 1023 × (C × 10-9/(L × 660) ("Copies/uL"), or [ 9.12 × 1011 × (C/(L × 660) ] (Copies/uL);

copies is the copy number; c is the standard plasmid DNA concentration, the unit ng.mu.L^-1(ii) a L is the standard plasmid DNA length in bp.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Sequence listing

<110> research institute for aquatic products in south China sea

<120> primer combination for detecting environment DNA of yellow fin spine porgy and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 27

<212> DNA

<213> COI COI P-F (Artificial sequence)

<400> 1

tggaggattc gggaattgat tagtacc 27

<210> 2

<211> 26

<212> DNA

<213> COI COI P-R (Artificial sequence)

<400> 2

gcggcaagaa ctggtagaga taagag 26

<210> 3

<211> 22

<212> DNA

<213> CO I QF1-F (Artificial sequence)

<400> 3

tgccgttcta cttctcttgt ct 22

<210> 4

<211> 24

<212> DNA

<213> CO I QF1-R (Artificial sequence)

<400> 4

gtgttggtaa agaatagggt ctcc 24

<210> 5

<211> 20

<212> DNA

<213> COI QF2-F (Artificial sequence)

<400> 5

actccggcta gatgaaggga 20

<210> 6

<211> 25

<212> DNA

<213> COI QF2-R (Artificial sequence)

<400> 6

acctcgccca tgcaggtgca tcagt 25

<210> 7

<211> 21

<212> DNA

<213> COI QF3-F (Artificial sequence)

<400> 7

ccggatggac agtttatccc c 21

<210> 8

<211> 22

<212> DNA

<213> COI QF3-R (Artificial sequence)

<400> 8

aaactccggc tagatgaagg ga 22

<210> 9

<211> 20

<212> DNA

<213> COI QF4-F (Artificial sequence)

<400> 9

agttcttgct gccggaatta 20

<210> 10

<211> 20

<212> DNA

<213> COI QF4-R (Artificial sequence)

<400> 10

ttcagggtgt ccgaagaatc 20

<210> 11

<211> 20

<212> DNA

<213> COI QF5-F (Artificial sequence)

<400> 11

gattcttcgg acaccctgaa 20

<210> 12

<211> 20

<212> DNA

<213> COI QF5-R (Artificial sequence)

<400> 12

atcatcgctc aaaccattcc 20

<210> 13

<211> 27

<212> DNA

<213> Probe (Artificial sequence)

<400> 13

ccagggttcg gtataatctc ccacatc 27

Claims

1. The primer combination for detecting the environmental DNA of the oplegnathus fasciatus is characterized by comprising a specific primer and a probe, wherein the nucleotide sequence of the specific primer is shown as SEQ ID NO: 9 and SEQ ID NO: 10, and the nucleotide sequences of the probes are respectively shown as SEQ ID NO: shown at 13.

2. The primer combination of claim 1, wherein the primer combination is used for detecting the environmental DNA of the oplegnathus fasciatus.

3. A kit for detecting environmental DNA of a yellow fin sea bream, comprising the specific primer of claim 1.

4. A fluorescence quantitative kit for detecting environmental DNA of a yellow fin sea bream, characterized by comprising the primer combination of claim 1.

5. A method for detecting yellow fin spine sea bream by eDNA technology, characterized in that the specific primer of claim 1 is used to perform PCR detection on the DNA of the living environment of the yellow fin spine sea bream, and whether the target species of the yellow fin spine sea bream exists in the water sample is determined and identified by detecting the existence of the strip.

6. The method for detecting yellow finny sea bream by eDNA technology as claimed in claim 5, wherein the PCR detection uses 50 μ L system including 2 XTaq Mix 25 μ L, less than 500ng DNA template 1-5 μ L, Primer F1 μ L, Primer R1 μ L, sterile water to 50 μ L.

7. The method for detecting yellow spine sea bream by eDNA technology according to claim 5, wherein the reaction procedure of PCR detection is 3 steps, specifically comprising the following steps: (1) pre-denaturation at 94 ℃ for 3 min; (2) denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and amplification for 35 cycles; (3) further extension was carried out at 72 ℃ for 5 min.

8. A PCR fluorescent quantitative method suitable for the eDNA of the yellow fin spine porgy, which is characterized in that the primer combination of claim 1 is used for carrying out fluorescent quantitative PCR on the DNA of the living environment of the yellow fin spine porgy, a standard curve between the concentration of the CO I gene plasmid standard of the yellow fin spine porgy and the Ct value is drawn, and finally the concentration of the DNA of the environment of the yellow fin spine porgy is determined according to the standard curve.

9. The method of claim 8, wherein the fluorescence quantitative PCR reaction system comprises 2 xqPCRMix 10. mu.L, 0.4. mu.L forward primer, 0.4. mu.L reverse primer, 0.4. mu.L probe, 2. mu.L template DNA, and 6.8. mu.L PCR water, wherein the fluorescence quantitative PCR reaction system comprises 20. mu.L PCR water.

10. The method of claim 8, wherein the fluorescent quantitative PCR amplification reaction procedure is a two-step method, and is specifically configured as follows: (1) pre-denaturation at 94 ℃ for 3 min; (2) denaturation at 94 ℃ for 5s, annealing and extension at 60 ℃ for 30s, and 40 cycles.