Primer combination and method for monitoring population distribution of spotted seal based on seawater sample

Abstract

The invention belongs to the technical field of nucleic acid detection, and relates to a primer combination and a method for monitoring population distribution of spotted seal based on a seawater sample. The primer combination comprises two pairs of specific primers, and forward primer sequences of the two pairs of primers are the same and are shown as SEQ ID NO.1 in a sequence table; a reverse primer sequence of the first pair of primers is as shown in SEQ ID NO. 2 in the sequence table; and the reverse primer sequence of the second pair of primers is shown as SEQ ID NO.3 in the sequence table. The method comprises the following steps: collecting a seawater sample of a monitoring area; extracting total DNA in the seawater sample; performing qPCR (quantitative polymerase chain reaction) amplification on the total DNA by using the primer combination; and judging the population distribution condition of the seal according to an amplification result. The method has the advantages of being easy and convenient to operate, high in sensitivity, high in specificity and the like, and an efficient and reliable molecular detection tool can be provided for protection, breeding and resource management of the seal.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology and relates to primer combinations and methods for monitoring the population distribution of spotted seals based on seawater samples. Background Technology

[0002] Spotted seal ( Phocalargha ) belongs to the order Carnivora, suborder Caniformia, family Phocidae, and genus Seal ( Phoca Genetic and ecological studies have shown that spotted seal populations in different distribution areas lack gene exchange, often forming independent evolutionary branches with unique genetic makeup, thus playing an important role in biodiversity conservation and management.

[0003] Traditional methods for monitoring the distribution of spotted seal populations mainly rely on manual patrols, acoustic monitoring, and drone monitoring. These methods are easily limited by factors such as weather, sunlight, and sea conditions, and have limited coverage and high costs.

[0004] In recent years, environmental DNA (eDNA) technology has been gradually applied to the distribution monitoring of aquatic organisms and has shown broad prospects. Among them, the macrobarcoding method can achieve simultaneous identification of multiple groups by amplification with universal primers and combined with high-throughput sequencing, which has advantages in community structure analysis and biodiversity assessment. However, for rare and endangered aquatic organisms, this method has limitations in practical applications: (1) The experimental process is complicated, requiring PCR amplification, library construction and sequencing, which takes a long time and is difficult to meet the monitoring needs of rapid response; (2) The universal primers of macrobarcoding often have amplification bias in complex environmental samples, and the signals of high-abundance species can easily mask the signals of low-abundance and rare species, thereby reducing the detection rate and stability of rare and endangered species. In contrast, the detection method based on species-specific primers can directly target the target DNA and has the advantages of non-destructive, high sensitivity, simple operation and low cost. It is especially suitable for environmental DNA samples with low concentration and severe degradation, and can realize accurate monitoring of rare and endangered species, thereby better serving the protection and management practice.

[0005] Currently, there is no species-specific environmental DNA molecular detection method for monitoring the distribution of spotted seal populations. This research gap, to some extent, limits the effective implementation of conservation and resource management efforts for spotted seals. Summary of the Invention

[0006] The purpose of this invention is to provide a primer combination and method for the rapid and specific identification of spotted seals under normal and extremely low environmental DNA concentration conditions. This method is accurate, highly sensitive, simple, fast, inexpensive, and requires minimal equipment, making it suitable for large-scale identification operations and showing promising application prospects.

[0007] The application provides a primer combination for monitoring population distribution of spotted seals based on seawater samples, and the primer combination comprises two pairs of specific primers.

[0008] The application provides a method for monitoring population distribution of spotted seals based on seawater samples. (1) collecting seawater samples in a monitoring area; (2) extracting total DNA from the seawater environmental samples; (3) performing qPCR amplification on the total DNA by using the primer combination; (4) determining the monitoring result according to the amplification result.

[0009] Preferably, the reaction system of the qPCR amplification comprises SYBR Green Pro Tag HS 10 μL, template DNA 1 μL, 0.4 μL of the forward primer and the reverse primer respectively, ddH2O 8.2 μL, and a total system 20 μL.

[0010] Preferably, the monitoring result determination method is that when the Ct value of the seawater sample is less than 34, the peak value of the melting curve of the first pair of primers is at 84℃ and the peak type is sharp and without sawtooth, or the peak value of the melting curve of the second pair of primers is at 85℃ and the peak type is sharp and without sawtooth, it is determined that the seawater sample contains environmental DNA of the spotted seal; when the detection results of the two pairs of primers are consistent, they can be verified with each other, so that the reliability of the monitoring result is further improved.

[0011] Preferably, the monitoring result determination further comprises performing gel electrophoresis on the amplification product and observing whether there is a target amplification band.

[0012] The application further provides a kit for monitoring population distribution of spotted seals based on seawater samples, and the kit comprises the primer combination.

[0013] The present application has the beneficial effects that: (1) the method not only realizes high-sensitivity amplification in low-concentration and seriously degraded seawater samples, significantly improves the efficiency and accuracy of monitoring the distribution of spotted seals in complex water bodies in the wild, but also significantly shortens the detection time (can be completed in a few hours), avoids the sequencing link, reduces the cost, and is convenient for large-scale application; (2) the method can efficiently and accurately detect whether there is a spotted seal in the target sea area, solves the problem that the traditional monitoring technology is easily restricted by environmental conditions (such as terrain, weather, and visibility) and human observation factors, and significantly reduces the monitoring cost; (3) based on wide-area sampling data, the method can clearly determine the main habitat range, distribution limit and possible migration path of the spotted seal, and provide decision basis for government departments to scientifically delineate and manage the protection zone. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Sequence comparison chart of the region of the designed species-specific forward primer for the D-Loop region of the mitochondrial genome of the spotted seal and the sequences of six closely related species; Figure 2 Agarose electrophoresis result chart of the PCR amplification products of two pairs of primers in Example 2 of the present application with spotted seal genomic DNA as the template at different concentrations; Figure 3 The amplification curves of the qPCR amplification products of two pairs of primers in Example 3 of the present application; wherein (a) is the amplification curve of the PL-1 primer; (b) is the amplification curve of the PL-2 primer; Figure 4 The melting curves of the qPCR amplification products of two pairs of primers in Example 3 of the present application; wherein (a) is the melting curve of the amplification product of the PL-1 primer; (b) is the melting curve of the amplification product of the PL-2 primer; Figure 5 Agarose electrophoresis verification chart of the amplification products of two pairs of primers in Example 3 of the present application. DETAILED DESCRIPTION

[0015] The present application will be further described below in conjunction with the drawings and specific application examples. It should be understood that these application examples are only used to illustrate the present application and not used to limit the scope of the present application. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the content taught by the present application, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

[0016] Example 1: Design of specific primers for monitoring the distribution of spotted seal population based on seawater samples The mitochondrial genome sequence of the spotted seal was downloaded from the NCBI database (NC_008430.1), and the specific primer for the spotted seal was designed using Primer Premier 5.0 according to the sequence alignment results of the D-Loop region of the mitochondrial genome of the spotted seal and six closely related species. Due to the high sequence similarity between the spotted seal and its closely related species in the D-Loop region, it is difficult to find a double-end segment that is highly species-specific and meets the requirements of primer sequence on both sides of the amplified fragment. Therefore, when designing, the region with two or more specific mutations or deletions at the 3' end was selected as the binding site of the forward primer. As shown in Table 1, based on the forward primer, two pairs of spotted seal-specific amplification primers located in the D-Loop region were designed using Primer Premier 5.0 software according to the annealing temperature and other primer design parameters. After comparison and screening, the optimal spotted seal-specific primer combination was determined as follows: Figure 1 The sequence of the first pair of primers PL-1 is as follows: Forward primer PL-F: 5'-AACACCAAGTTCTAAAGC-3', SEQ ID NO. 1; Reverse primer PL-1R: 5'-GGAGCGAGGTTTAGGTAC-3', SEQ ID NO. 2. The length of the amplified product is 121 bp.

[0017] The sequence of the second pair of primers PL-2 is as follows:

[0018] Forward primer PL-F: 5'-AACACCAAGTTCTAAAGC-3', SEQ ID NO. 1; Reverse primer PL-2R: 5'-GATGCCAGGTATAGTTCC-3', SEQ ID NO. 3. The length of the amplified product is 169 bp.

[0019] Among them, PL-1 and PL-2 share the same forward primer sequence PL-F, only differ in the reverse primer, and thus different lengths of target fragments are amplified respectively. This design not only ensures the sensitivity and specificity of the detection, but also enables mutual verification during the detection process, improving the reliability and applicability of the detection results.

[0020] Example 2: Sensitivity verification of specific primers

[0021] ​(1) Take muscle tissue samples (20 mg each) from 4 spotted seals and extract their genomic DNA using the phenol-chloroform method. The template concentrations of the resulting DNA stock solution were 267.3 ng / μL, 334.6 ng / μL, 329.1 ng / μL and 303.4 ng / μL, respectively.

[0022] (2) The concentrations of the above DNA samples were diluted to 1 / 10,000 and 1 / 100,000 respectively as DNA templates, and PCR amplification was performed using specific primers PL-1 and PL-2 respectively.

[0023] The PCR amplification system consisted of a total volume of 25 μL, including 12.5 μL of PCR Mix, 1 μL of DNA template, 0.5 μL each of 10 μM / μL forward and reverse primers, and 10.5 μL of sterile double-distilled water.

[0024] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 50 s, 35 cycles, and a final extension at 72℃ for 5 min.

[0025] (3) Take 2 μL of PCR amplification product for agarose gel electrophoresis. The gel concentration is 1.5%; the electrophoresis buffer is 1×TAE, the voltage does not exceed 5V / cm, and the electrophoresis time is 30-40 min. The amplified bands are detected by UV gel imaging.

[0026] like Figure 2 As shown, M is the DL2000 DNA Marker. A1-A4 and B1-B4 represent the bands amplified using primers PL-1 and PL-2 when the spotted seal DNA stock solution was diluted 1 / 10,000 (corresponding to concentrations of 0.027 ng / μL, 0.033 ng / μL, 0.033 ng / μL, and 0.030 ng / μL, respectively). A5–A8 and B5–B8 represent the amplification results when the spotted seal DNA stock solution was diluted 1 / 100,000 (corresponding to concentrations of 0.002 ng / μL, 0.003 ng / μL, 0.003 ng / μL, and 0.003 ng / μL, respectively). The results show clear and specific amplification bands at 121 bp and 169 bp, indicating that the primers still have high sensitivity and specificity under extremely low template concentration conditions.

[0027] Example 3: The above-mentioned specific primers were used to monitor the population distribution of spotted seals. The specific method is as follows: (1) The surface seawater was collected in 2.5L organic glass water sampler from the large-scale breeding pool, small-scale breeding pool and seawater outside the seal colony of Qingdao Underwater World, and two parallel samples were set. After collection, the seawater was filtered with sterilized gauze to remove large particles of silt, and then the suspended particles were intercepted on 0.2μm mixed cellulose filter membrane by sand core filtering device. The filter membrane was folded and stored in -80℃ ultra-low temperature refrigerator.

[0028] (2) The filter membrane was taken out, cut into pieces with sterile scissors, and then put into an EP tube. Glass beads were added into the EP tube, and the beads were ground with a bead mill tissue grinder at a speed of 2500rpm until the whole homogenate was obtained. After soaking in BS buffer, the homogenate was centrifuged at 8000rpm for 10min, and the precipitate was extracted by DNeasy Blood&Tissue Kit.

[0029] (3) The specific primers PL-1 and PL-2 were used, and the SYBR Green Pro Tag HS kit was used for amplification on Rotor-Gene Q real-time fluorescent quantitative PCR analyzer. The reaction system was 20μL, including: SYBR Green Pro Tag HS 10μL, template DNA 1μL, forward primer and reverse primer 0.4μL each, and ddH2O 8.2μL. The amplification conditions were: 95℃ pre-denaturation for 30s, 95℃ denaturation for 5s, 60℃ annealing for 30s, a total of 40 cycles; then melt curve analysis was carried out, the temperature program was increased from 50℃ to 99℃ at a gradient of 1℃, and the fluorescence signal change was continuously monitored, so as to obtain the melting curve of the amplification product.

[0030] As shown in Figure 3 (a) is the amplification curve of PL-1 primer, L1 and L2 are the amplification curves of the large-scale breeding pool of seal colony, L3 and L4 are the amplification curves of the small-scale breeding pool of seal colony, and L5 and L6 are the amplification curves of seawater sample in the seawater discharge area outside the seal colony; (b) is the amplification curve of PL-2, L7 and L8 are the amplification curves of the large-scale breeding pool of seal colony, L9 and L10 are the amplification curves of the small-scale breeding pool of seal colony, and L11 and L12 are the amplification curves of seawater sample in the seawater discharge area outside the seal colony; there are differences between the amplification curves of the three groups of water samples, which represent that the primers can work normally and the method can distinguish the content of environmental DNA.

[0031] (4) The method for judging the monitoring results is: when the Ct value of seawater sample is less than 34, the peak value of melting curve of PL-1 primer is at 84℃ and the peak type is sharp without jagged, or the peak value of melting curve of PL-2 primer is at 85℃ and the peak type is sharp without jagged, it can be judged that there is environmental DNA of spotted seal in the seawater sample; when the detection results of the two pairs of primers are consistent, they can be verified with each other, so as to further improve the reliability of the monitoring results.

[0032] As shown in Figure 4 Fig. 1 shows the melting curves of the amplification products of the primers, wherein (a) is the melting curve of the amplification product of the primer PL-1, L1 and L2 are the melting curves of the water samples in the large-scale breeding pool of the seal pavilion, L3 and L4 are the melting curves of the water samples in the small-scale breeding pool of the seal pavilion, L5 and L6 are the melting curves of the water samples in the seawater discharge area outside the pavilion, and the peak values are all 84℃; (b) is the melting curve of the amplification product of the primer PL-2, L7 and L8 are the melting curves of the water samples in the large-scale breeding pool of the seal pavilion, L9 and L10 are the melting curves of the water samples in the small-scale breeding pool of the seal pavilion, L11 and L12 are the melting curves of the water samples in the seawater discharge area outside the pavilion, and the peak values are all 85℃.

[0033] (5) 2 μL of the amplification product is subjected to 1.5% agarose gel electrophoresis (1xTAE buffer, voltage ≤ 5 V / cm, 30-40 min), and is detected by an ultraviolet gel imaging instrument.

[0034] As shown in Figure 5 Fig. 2 shows the electrophoresis results of the amplification products of the primers, wherein M is a DL2000 DNA marker, D1 and D2 are the amplification bands of the primer PL-1 of the water sample in the large-scale breeding pool, D3 and D4 are the amplification bands of the primer PL-2 of the water sample in the large-scale breeding pool, E1 and E2 are the amplification bands of the primer PL-1 of the water sample in the small-scale breeding pool, E3 and E4 are the amplification bands of the primer PL-2 of the water sample in the small-scale breeding pool, F1 and F2 are the amplification bands of the primer PL-1 of the water sample in the seawater discharge area outside the pavilion, and F3 and F4 are the amplification bands of the primer PL-2 of the water sample in the seawater discharge area outside the pavilion. The results all show specific bands corresponding to the target fragments.

[0035] In summary, the method provided by the present application is suitable for effectively monitoring the population distribution of the spotted seal based on seawater samples, has the advantages of simple operation, high sensitivity and strong specificity, and can provide an efficient molecular tool for the protection, breeding and resource management of the spotted seal.

Claims

1. A primer combination for monitoring the population distribution of the spotted seal based on a seawater sample, characterized in that: The primer combination comprises two pairs of specific primers, the forward primers of the two pairs of primers have the same sequence as shown in the sequence table SEQ ID NO. 1, the reverse primer sequence of the first pair of primers is shown in the sequence table SEQ ID NO. 2, and the reverse primer sequence of the second pair of primers is shown in the sequence table SEQ ID NO.

3.

2. A method for monitoring the distribution of a population of ringed seals based on seawater samples, characterized in that The method comprises the following steps: (1) collecting seawater samples in a monitoring area; (2) extracting total DNA from the seawater environmental samples; (3) performing qPCR amplification on the total DNA by using the primer combination in claim 1; (4) determining the population distribution of the spotted seal according to the amplification results.

3. The method of claim 2, wherein, The reaction system of the qPCR amplification comprises: SYBRGreen Pro Tag HS 10 μL, template DNA 1 μL, forward primer and reverse primer each 0.4 μL, ddH2O 8.2 μL, and the total system 20 μL.

4. The method of claim 2, wherein, The amplification result determination method is: when the Ct value of the seawater sample is less than 34, and the peak value of the melting curve of the first pair of primers is at 84℃, the peak type is sharp without sawtooth, or the peak value of the melting curve of the second pair of primers is at 85℃, the peak type is sharp without sawtooth, it is determined that the seawater sample contains environmental DNA of the spotted seal; when the detection results of the two pairs of primers are consistent, it is determined that the monitoring result is accurate and reliable.

5. The method of claim 4, wherein, The amplification result determination further comprises: performing gel electrophoresis detection on the amplification product to observe whether there is a specific band consistent with the expected size; when both pairs of primers amplify the expected band, it is determined that the monitoring result is accurate and reliable.

6. A kit for monitoring the distribution of a population of ringed seals based on seawater samples, characterized in that: The kit comprises the primer combination in claim 1.

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