Environmental DNA ampullaria gigas PCR detection method and application thereof

Abstract

The invention discloses an environmental DNA (Deoxyribose Nucleic Acid) ampullaria gigas PCR (Polymerase Chain Reaction) detection method and application thereof, according to the method for detecting the environmental DNA ampullaria gigas, the detection limit in a water body is as low as 0.4 copies / mu L, only the target species ampullaria gigas is successfully amplified, near-source species (such as field snails and the like) cannot be amplified, and the method has relatively high detection sensitivity and specificity.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular detection, and in particular relates to an environmental DNA Pomacea canaliculata PCR detection method and application thereof. Background Art

[0002] There are many morphologically similar species in the genus Pomacea, which is the most difficult genus to identify in the family Pomacea. For example, Pomacea canaliculata is similar to Pomacea insularum, Pomacealineata, Pomacea dolioides, Pomacea haustrum, Pomacea bridgesi and Pomacea paludosa. They can be distinguished mainly by morphological characteristics such as the color of the eggs, the color or color bands of the shells, and the angle between the whorls.

[0003] Pomacea canaliculata has been included in the supplementary list of agricultural quarantine pests. Pomacea canaliculata has been found to cause harm in rivers, ditches, rice fields, and aquatic crops such as water chestnuts, water caltrops, lotus roots, and wild rice stems. Therefore, it is necessary to conduct a risk analysis of the introduction and spread of Pomacea canaliculata. Establishing a qPCR method that can simultaneously detect all Pomacea canaliculata species under the genus Pomacea canaliculata and provide technical support for detecting the content of Pomacea canaliculata in the environment is a technical problem to be solved. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0005] As one aspect of the present invention, the present invention provides an environmental DNA Pomacea canaliculata PCR detection method, which includes PCR detection primers, and the PCR detection primer sequence is: upstream primer sequence: Downstream primer sequence:

[0006] As a preferred embodiment of the environmental DNA Pomacea genus PCR detection method described in the present invention: the Pomacea genus includes golden apple snail (Pomacea bridgesi), small tube apple snail (Pomacea canaliculata), bottle-shaped apple snail (Pomacea doliodes), and spotted apple snail (Pomacea maculata).

[0007] As a preferred embodiment of the environmental DNA Pomacea canaliculata PCR detection method of the present invention: the annealing temperature of the PCR detection is 51-57°C.

[0008] As a preferred embodiment of the environmental DNA Pomacea canaliculata PCR detection method of the present invention: the PCR detection includes ordinary PCR detection or fluorescent quantitative PCR detection.

[0009] As a preferred scheme of the environmental DNA Pomacea canaliculata PCR detection method described in the present invention: the PCR detection program is: 50°C, 2 min; 95°C, 2 min; 95°C, 15 s; 57°C, 1 min, 40 cycles.

[0010] Beneficial effects of the present invention: The method for detecting environmental DNA Pomacea canaliculata of the present invention has a detection limit as low as 0.4 copies / μL in water bodies, and only the target species Pomacea canaliculata is successfully amplified, while closely related species (such as Chinese round field snails, etc.) cannot be amplified, and has high detection sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0012] Figure 1 It is a standard curve graph.

[0013] Figure 2 This is a diagram of the sampling point layout.

[0014] Figure 3 This is a graph showing the results of primer specificity verification and reaction condition optimization.

[0015] Figure 4 This is the environmental sample test results. DETAILED DESCRIPTION

[0016] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0017] Detection of Pomacea canaliculata species by PCR method:

[0018] 1) The first pair of primers: Upstream primer sequence: Downstream primer sequence:

[0019] Standard plasmid amplification sequence:

[0020]

[0021] 2) Universal primer: Upstream primer sequence: Downstream primer sequence:

[0022] Embodiment 1:

[0023] Draw a standard curve:

[0024] The product copy number calculation formula is as follows:

[0025] Copies = [(6.02 × 10 23 )×(C×10 -9 ) / (L×660)]×V

[0026] Copies is the copy number; C is the standard plasmid DNA concentration, unit ng·μL -1 ; L is the length of standard plasmid DNA, unit bp; V is the volume of standard plasmid DNA in real-time fluorescence q PCR system.

[0027] The standard plasmid was diluted to 1Copies / μl, 10Copies / μl, 10 2 Copies / μl, 10 3 Copies / μl, 10 4 Copies / μl, 10 5 Copies / μl, 10 6 Copies / μl, 10 7 Copies / μl. Each sample was replicated 3 times, and each qPCR plate needed to select 5 qPCR standard plasmid concentration gradients to make the standard curve.

[0028] The PCR amplification process is as follows:

[0029] (1) Add 10 μL PCR buffer, 1.5 μL upstream primer, 1.5 μL downstream primer, 5 μL ddH2O, and 2 μL standard plasmid to a PCR amplification tube.

[0030] (2) Turn on the PCR instrument and set the constant temperature section in the detection program settings: 50°C, 2 min; 95°C, 2 min; cycle section: 95°C, 15 s; 57°C, 1 min; 40 cycles.

[0031] After the program is finished, export the standard curve. Figure 1 From the results, it can be seen that the primer standard curve fitting effect is good. It has good amplification consistency and R 2 >0.99, the result is highly reliable.

[0032] Embodiment 2:

[0033] Environmental sample collection: Based on the two lake tributaries in Nanjing, six points were set up in the Xuanwu Lake tributary near Huaxin Road in Xuanwu District and the Nanhu Lake tributary near Nanhu Road in Jianye District. 5L of water was collected at each point, and the environmental DNA integrated enrichment kit was used with a water environment DNA filter to complete sample enrichment. The sampling details are as follows:

[0034] Sampling points were set up in the river near Huaxin Road, Xuanwu District (118.8373802° east longitude, 32.0914992° north latitude) and Nanhu Road, Jianye District (118.7600257° east longitude, 32.0328263° north latitude), with a total of 12 sampling points. The point map is shown in Figure 2 shown.

[0035] Primer specificity verification and reaction condition optimization:

[0036] The tissue DNA of Pomacea canaliculata, Pomacea edulis, and Pomacea maculatum were used as positive samples, the tissue DNA of Chinese round field snail and moon snail were used as negative samples, and DEPC water was used as blank control. The specificity of primers was detected by 2.0% agarose gel electrophoresis of PCR products.

[0037] Add 15 μL Taq mix, 1 μL upstream primer (the first pair of primers or universal primers), 1 μL downstream primer (the first pair of primers or universal primers), 11 μL ddH2O and 2 μL of each group of DNA samples to the PCR amplification tube in sequence.

[0038] Turn on the PCR instrument and set the program settings as follows: pre-denaturation: 95°C, 3 min; cycle: 95°C, 15 s; primer annealing temperature (48°C, 51°C, 54°C, 57°C), 20 s; 72°C, 40 s; 40 cycles; final extension: 72°C, 5 min; store at 4°C. Figure 3 As shown in the figure, the first pair of primers only amplified the genus Pomacea, but not the negative samples such as the closely related species Chinese round field snail, moonlight snail, and enzyme-free water, and the primers had strong specificity. However, the universal primers amplified bands for the closely related species Chinese round field snail and moonlight snail, and the primers had poor specificity.

[0039] Environmental sample testing: Pomacea canaliculata 10 5 ~10 1 Copies / μl standard plasmid was used as a standard curve, water samples S1 to S12 were used as positive environmental samples, tissue DNA of Chinese round field snails and moon snails, and enzyme-free water were used as negative control samples, and the first pair of primers were used to perform q PCR quantitative detection on environmental samples, with 3 replicates for each sample.

[0040] Add 10 μL of PCR buffer, 1.5 μL of upstream primers (the first pair of primers), 1.5 μL of downstream primers (the first pair of primers), 5 μL of ddH2O, and 2 μL of standard plasmid or environmental sample DNA to the PCR amplification tube in sequence.

[0041] Turn on the PCR instrument and set the constant temperature section in the detection program setting: 50℃, 2min; 95℃, 2min; cycle section: 95℃, 15s; 57℃, 1min; 40 cycles.

[0042]

[0043]

[0044] The experimental results are as follows Figure 4 As shown in the figure, we can see that all environmental samples containing the genus Pomacea canaliculata were detected, and the average water concentration was between 4.20 and 27.07 Copies / μl; no negative control samples were detected, indicating that the primer detection results were accurate.

[0045] In the present invention, the detection limit of each environmental sample is as low as 0.4 Copies / μl.

[0046] Comparative Example 1:

[0047] The tissue DNA of golden apple snail (Pomacea bridgesi), small tube apple snail (Pomacea canaliculata), bottle-shaped apple snail (Pomacea doliodes), and spotted apple snail (Pomacea maculata) were used as positive samples, and the moonlight snail (Marisa cornuarietis), bottle snail (Pila globosa), Chinese round field snail (Cipangopaludinachinensis), and long spiral round field snail (Cipangopaludina longispira) were used as negative controls to investigate the detection specificity of each set of primers for the genus Pomacea or related species, as shown in Table 1.

[0048] Table 1

[0049]

[0050]

[0051] As can be seen from Table 1, the first pair of primers can detect the genus Pomacea, while other closely related species cannot be detected, indicating that the first pair of primers has the best detection specificity.

[0052] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A PCR detection method for environmental DNA Pomacea canaliculata, characterized in that: The method comprises PCR detection primers, wherein the sequences of the PCR detection primers are: upstream primer sequence: 5′-CACTGAATGAAATGTAATTTCTTCTA-3′; and downstream primer sequence: 5′-TCATTCTAAGGCAGTAACTAAATGTAGT-3′.

2. The environmental DNA PCR detection method for Pomacea canaliculata according to claim 1, characterized in that: The genus Pomacea includes golden apple snail (Pomacea bridgesi), small tube apple snail (Pomacea canaliculata), bottle-shaped apple snail (Pomacea doliodes), and spotted apple snail (Pomacea maculata).

3. The environmental DNA PCR detection method for Pomacea canaliculata according to claim 1 or 2, characterized in that: The annealing temperature of the PCR detection is 51-57°C.

4. The environmental DNA PCR detection method for Pomacea canaliculata according to claim 1 or 2, characterized in that: The PCR detection includes common PCR detection or fluorescent quantitative PCR detection.

5. The environmental DNA PCR detection method for Pomacea canaliculata according to claim 1 or 2, characterized in that: The PCR detection program is: 50°C, 2 min; 95°C, 2 min; 95°C, 15 s; 57°C, 1 min, 40 cycles.

6. Use of the environmental DNA Pomacea canaliculata PCR detection method according to claim 1 in the identification of Pomacea canaliculata.

7. Use of the environmental DNA Pomacea canaliculata PCR detection method according to claim 1 in the preparation of a Pomacea canaliculata detection kit, characterized in that: The kit comprises the PCR detection primers.

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