Enzymatic cleavage site for discriminating between l. lanka and l. aborica and its use

Abstract

This invention provides an enzyme restriction site for distinguishing between *Pomacea canaliculata* and *Pomacea cryptica*, and its application, belonging to the field of molecular biology detection technology. The enzyme restriction site is based on the base difference between *Pomacea canaliculata* and *Pomacea cryptica*; the sequence of *Pomacea canaliculata* is ggtgcc, and the sequence of *Pomacea cryptica* is Agtgcc. The enzyme restriction site is the BshNI restriction site, and the recognition site is ggtgcc. Using this enzyme restriction site, *Pomacea canaliculata* and *Pomacea cryptica* can be distinguished. This invention utilizes the specific base difference in the mitochondrial COI gene sequences of the two species. Barcoding primers are used to amplify the conserved COI fragment, and the BshNI restriction site (recognition site ggtgcc) is obtained after screening. Based on this, the BshNI restriction endonuclease is used for enzyme digestion, resulting in distinctly different enzyme digestion electrophoretic bands for the two species, thereby achieving rapid, accurate, and batch identification.

Description

Technical Field

[0001] This invention belongs to the field of molecular identification of invasive alien organisms, quarantine of aquatic organisms and molecular biological detection technology, and relates to an enzyme cleavage site for identifying *Pomacea canaliculata* and *Pomacea cryptica* and its application. Background Technology

[0002] Small golden apple snail ( Pomacea canaliculata Golden apple snail (Pomacea canaliculata) is a major invasive aquatic organism in my country, damaging crops such as rice and vegetables, and is also a major intermediate host of Angiostrongylus cantonensis. Pomacea occulta Because they are closely related species, their shell morphology, size, and lifestyle are highly similar, making morphological identification extremely easy to confuse. Traditional classification relies on characteristics such as shell height, width, whorls, and aperture morphology, which are highly variable due to factors such as growth environment, nutritional conditions, and developmental age; juvenile snails, egg masses, and fragmented tissue samples cannot be accurately identified by morphology alone. Conventional DNA barcoding sequencing is costly and time-consuming, making it unsuitable for large-scale sample screening at the grassroots level. PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) relies on differences in gene sequence bases. Through PCR amplification, restriction endonuclease digestion, and electrophoretic typing, it can achieve rapid and visual identification of closely related species. It is simple to operate, low in cost, and has good reproducibility. Currently, there is no standardized COI-PCR-RFLP identification method for *Pomacea canaliculata* and *Pomacea cryptica*, making the establishment of a rapid molecular identification technology of significant application value. Summary of the Invention

[0003] To address the aforementioned problems, this invention overcomes the shortcomings of existing methods, such as difficulty in morphological identification, high sequencing detection costs, and long processing times. It provides an enzyme-digested gene for identifying *Pomacea canaliculata* and *Pomacea cryptica*, and a rapid PCR-RFLP method for identifying the mitochondrial COI gene of these two species using this gene. This invention utilizes the specific base differences in the mitochondrial COI gene sequences of the two species, amplifies the conserved COI fragment using universal barcoding primers, and performs restriction endonuclease digestion. The two species produce distinctly different enzyme digestion electrophoresis bands, achieving rapid, accurate, and batch identification.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An enzyme cleavage site for distinguishing between *Pomacea canaliculata* and *Pomacea cryptica*, wherein the enzyme cleavage site is a base difference between the two snippets, with the sequence of *Pomacea canaliculata* being ggtgcc and the sequence of *Pomacea cryptica* being Agtgcc; the enzyme cleavage site is a BshNI cleavage site, and the recognition site is ggtgcc. Using the enzyme cleavage sites, *Pomacea canaliculata* and *Pomacea cryptica* can be distinguished.

[0005] A rapid PCR-RFLP method for identifying the mitochondrial COI gene of *Pomacea canaliculata* and *Pomacea cryptica*, comprising the following steps: 1) Extract genomic DNA from the sample to be tested, dilute it, and use it as template DNA; 2) Perform PCR amplification using template DNA to obtain PCR amplification products; 3) Based on the above restriction sites, the restriction endonuclease BshNI was selected to perform isothermal restriction digestion on the PCR amplification products; the recognition site of restriction endonuclease BshNI is ggtgcc. 4) The obtained enzyme digestion products are detected by gel electrophoresis to determine the type of sample to be tested; In the gel electrophoresis results of the enzyme digestion products, the sample tested was *Pomacea canaliculata* when two bands were generated, and the sample tested was *Pomacea cryptica* when one band was generated.

[0006] Furthermore, the enzyme digestion reaction system includes PCR amplification products, 10× enzyme digestion buffer, restriction endonuclease BshNI, and ddH2O.

[0007] Furthermore, the enzyme digestion reaction system includes 10 μL of PCR amplification product, 2 μL of 10× enzyme digestion buffer, 1 μL of restriction endonuclease BshNI, and 7 μL of ddH2O.

[0008] Furthermore, the enzyme digestion reaction was carried out at a constant temperature of 65°C for 2 hours.

[0009] Furthermore, the PCR amplification system includes template DNA, upstream and downstream primers, 10×Taq buffer, dNTPs, MgCl2, Taq enzyme, and ddH2O; The upstream and downstream primers are as follows: Upstream primer LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3'; Downstream primer HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'.

[0010] Furthermore, the PCR amplification system includes 1 μL template DNA, 1 μL each of 10 μmol / L upstream and downstream primers, 2.5 μL 10×Taq buffer, 2 μL 2.5 mmol / L dNTPs, 1 μL 25 mmol / L MgCl2, 0.25 μL 5 U / μL Taq enzyme, and ddH2O to a final volume of 25 μL. The upstream and downstream primers are as follows: Upstream primer LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3'; Downstream primer HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'.

[0011] Furthermore, the PCR amplification program was as follows: pre-denaturation at 94℃ for 5 min; 35 cycles of 94℃ for 30 s, 50℃ for 30 s, and 72℃ for 50 s; final extension at 72℃ for 10 min; and storage at 4℃.

[0012] Furthermore, gel electrophoresis was performed using a 1.5% agarose gel at a voltage of 120V for 25-30 minutes.

[0013] The beneficial effects of the enzyme cleavage sites of this invention for distinguishing between *Pomacea canaliculata* and *Pomacea cryptica* and their applications are as follows: To address the challenges of the highly similar morphology between *Pomacea canaliculata* and *Pomacea cryptica*, the difficulty in traditional morphological identification, reliance on experience, and the inability to accurately distinguish juveniles and remains, this invention utilizes the specific base differences in the mitochondrial COI gene sequences of the two species. Barcoding primers are used to amplify conserved COI fragments, and BshNI restriction sites (recognition site is ggtgcc) are obtained through screening. Based on this, a specific restriction endonuclease BshNI is used for digestion. After agarose gel electrophoresis, distinctly different restriction bands are produced between the two species. Rapid, accurate, and batch identification is achieved based on the differences in the number and size of the restriction bands between the two species. This invention uses COI barcode primers, eliminating the need to design species-specific primers, and is highly versatile and low-cost; The detection process of this invention is simple and can be completed in three steps: PCR, enzyme digestion and electrophoresis. Results can be obtained in 4-6 hours, which is much faster than sequencing identification. This invention is not limited by the developmental age of the sample, shell defects, or tissue fragments, and is applicable to various field and quarantine samples. This invention provides intuitive and simple band differences for interpretation, requiring no bioinformatics analysis, and can be widely used in grassroots plant protection and aquatic quarantine. This invention enables large-scale parallel detection and is suitable for baseline population surveys of golden apple snails, invasion monitoring, evaluation of control effectiveness, and tracing of parasite hosts. This invention is simple to operate, time-saving, highly specific, and reproducible. It is not affected by the developmental stage, integrity, or environmental phenotype of the sample, and can be applied to aquatic quarantine, invasive species monitoring, field population surveys, and other fields. Attached Figure Description

[0014] Figure 1 This is a diagram of the COI gene restriction sites of *Pomacea canaliculata* and *Pomacea cryptica* in Example 1 of this invention; Figure 2This is a PCR-RFLP electrophoresis pattern diagram of the COI genes of *Pomacea canaliculata* and *Pomacea cryptica* in Example 2 of this invention; M represents Marker; 1-1 represents the electrophoresis result of the PCR product of the *Pomacea canaliculata* COI fragment; 1-2 represents the electrophoresis result of the enzyme digestion product of the *Pomacea canaliculata* COI amplified fragment; 2-1 represents the electrophoresis result of the PCR product of the *Pomacea cryptica* COI fragment; 2-2 represents the electrophoresis result of the enzyme digestion product of the *Pomacea cryptica* COI fragment. Figure 3 These are gel images of enzyme digestion identification of Pomacea canaliculata from different regions in Example 3 of this invention; where M represents Marker, numbers 1 to 8 represent Pomacea canaliculata corresponding to numbers 1 to 8 in Table 1, and numbers 9 to 16 represent Pomacea canaliculata corresponding to numbers 9 to 16 in Table 1. Detailed Implementation

[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it.

[0016] Example 1: Design of restriction enzyme sites for differentiating between *Pomacea canaliculata* and *Pomacea cryptica* 1) Extract genomic DNA from *Pomacea canaliculata* and *Pomacea cryptica*. Samples of *Pomacea canaliculata* and *Pomacea cryptica* were collected in the field and stored in a -20°C refrigerator.

[0017] Take an appropriate amount of tissue block, add 1 ml of 2% CTAB extraction buffer (2% CTAB formula: 100 mmol / L Tris-HCl pH 7.0, 20 mmol / L EDTA, 1.4 mol / L NaCl) and homogenize thoroughly. Then transfer 700 μL of homogenate to a new centrifuge tube and incubate at 65°C for 45 min. Add 700 μL of a chloroform and isoamyl alcohol mixture with a volume ratio of 24:1, and shake up and down for 2-3 minutes; Centrifuge at 12000×g for 10 min; Take about 500 μL of supernatant and transfer it to a new centrifuge tube. Add 1 / 10 volume of NaAc (3 mol / L, pH 5.2) and mix by inverting the tube. Add twice the volume of ice-cold anhydrous ethanol, mix by inverting the container; let stand at -20°C for at least 1 hour. Centrifuge at 12000×g for 10 min, discard the supernatant; wash the precipitate twice with 70% ethanol. After the ethanol has evaporated, add 50-100 μL of TE buffer (containing 50 μg / ml RNase) and dissolve thoroughly. Electrophoresis is used to detect the integrity of genomic DNA; The concentration of genomic DNA was detected using nanodrop; Store at -20℃ or -80℃.

[0018] 2) Obtaining restriction enzyme sites Genomic DNA from *Pomacea canaliculata* and *Pomacea cryptica* was diluted 10-fold and used as template DNA for PCR amplification to obtain the target fragment of the COI gene. The primer sequences for the COI gene are as follows: Upstream primer LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3'; Downstream primer HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'; The total volume of the PCR amplification system was 25 μL, including: 1 μL template DNA, 1 μL each of 10 μmol / L forward and reverse primers, 2.5 μL 10×Taq buffer, 2 μL 2.5 mmol / L dNTPs, 1 μL 25 mmol / L MgCl2, 0.25 μL 5 U / μL Taq enzyme, and ddH2O to bring the total volume to 25 μL.

[0019] The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ for 30 s, 50℃ for 30 s, 72℃ for 50 s, 35 cycles; 72℃ final extension for 10 min; and storage at 4℃.

[0020] The PCR amplification products were divided into two portions. One portion was sent to Qingke Biotechnology for Sanger sequencing. After sequencing, the COI genomes of *Pomacea canaliculata* and *Pomacea cryptica* were aligned using SnapGene software. Differential restriction enzyme sites in the COI sequences of the two species were screened. Analysis revealed a base difference at 200bp between *Pomacea canaliculata* and *Pomacea cryptica*. The sequence from *Pomacea canaliculata* was ggtgcc, while the sequence from *Pomacea cryptica* was Aggtgcc. This differential site is the BshNI restriction enzyme site. (Details are as follows...) Figure 1 As shown.

[0021] The COI gene sequence of *Pomacea canaliculata* is shown in SEQ ID NO: 1, and the COI gene sequence of *Pomacea cryptica* is shown in SEQ ID NO: 2.

[0022] Example 2: A rapid PCR-RFLP method for identifying the mitochondrial COI gene of *Pomacea canaliculata* and *Pomacea cryptica*. 1) Extract genomic DNA from *Pomacea canaliculata* and *Pomacea cryptica*. Genomic DNA was extracted from *Pomacea canaliculata* and *Pomacea cryptica* according to the method in Example 1, and diluted 10-fold as the corresponding template DNA.

[0023] 2) PCR amplification Template DNA from *Pomacea canaliculata* and *Pomacea cryptica* was amplified by PCR to obtain PCR amplification products. The primer sequences for the COI gene are as follows: Upstream primer LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3'; Downstream primer HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'; The total volume of the PCR amplification system was 25 μL, including: 1 μL template DNA, 1 μL each of 10 μmol / L forward and reverse primers, 2.5 μL 10×Taq buffer, 2 μL 2.5 mmol / L dNTPs, 1 μL 25 mmol / L MgCl2, 0.25 μL 5 U / μL Taq enzyme, and ddH2O to bring the total volume to 25 μL.

[0024] The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ for 30 s, 50℃ for 30 s, 72℃ for 50 s, 35 cycles; 72℃ final extension for 10 min; and storage at 4℃.

[0025] 3) Rapid identification by PCR-RFLP Based on the BshNI restriction site (recognition site is ggtgcc) obtained in Example 1, the restriction endonuclease BshNI was used to perform an isothermal restriction digestion reaction on the PCR amplification product. The total volume of the enzyme digestion reaction system was 20 μL, including: 10 μL of PCR amplification product, 2 μL of 10× enzyme digestion buffer, 1 μL of restriction endonuclease BshNI, and 7 μL of ddH2O; the enzyme digestion was carried out at 65℃ for 2 h.

[0026] 4) Gel electrophoresis The obtained enzyme digestion products and PCR amplification products were subjected to agarose gel electrophoresis on a 1.5% agarose gel at 120V for 25-30 min. After staining and imaging, the band patterns were observed using a gel imaging system. Figure 2 As shown.

[0027] according to Figure 2The number and size of electrophoretic bands were used to distinguish between *Pomacea canaliculata* and *Pomacea cryptica*. The criteria for differentiation were as follows: *Pomacea canaliculata* and *Pomacea cryptica* both produced two bands of identical size after PCR amplification, making it impossible to directly distinguish between the two species based solely on the size of the PCR product; however, *Pomacea canaliculata* produced two bands after enzyme digestion, while *Pomacea cryptica* produced one band. The number of bands after enzyme digestion directly identified the species. In other words, in the gel electrophoresis results of the enzyme digestion products, a sample producing two bands indicates *Pomacea canaliculata*, and a sample producing one band indicates *Pomacea cryptica*.

[0028] Following the above experimental steps, parallel experiments were conducted on known samples of *Pomacea canaliculata* and *Pomacea cryptica*, with three repetitions. The electrophoretic band characteristics were stable and reproducible, demonstrating the ability to reliably distinguish between the two closely related species of *Pomacea canaliculata*, and can be used for actual sample detection.

[0029] Example 3: Identification of Pomacea canaliculata results from different regions DNA was extracted from *Pomacea canaliculata* samples collected from different regions of Hainan according to the method described in Example 2 (Table 1), and enzyme digestion verification was performed sequentially according to the method described in Example 2. The enzyme digestion verification results are shown in [Table 1]. Figure 3 This study showed that different individuals of the same species can maintain a high degree of accuracy and stability.

[0030] Table 1. Verification results of two species of golden apple snail in different regions.

[0031] The results of Examples 2 and 3 demonstrate that, based on the BshNI restriction site (recognition site is ggtgcc) obtained in Example 1, the PCR amplification products of COI from *Pomacea canaliculata* and *Pomacea canaliculata* can be accurately and quickly distinguished from two similar *Pomacea canaliculata* species in China by using the restriction endonuclease BshNI for enzyme digestion and identification.

[0032] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. Those skilled in the art can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An enzyme cleavage site for discriminating between Pomacea canaliculata and Pomacea parthenopea, characterized in that, The enzyme cleavage site is due to the base difference between *Pomacea canaliculata* and *Pomacea cryptica*, with the sequence of *Pomacea canaliculata* being ggtgcc and the sequence of *Pomacea cryptica* being Agtgcc; the enzyme cleavage site is the BshNI cleavage site, and the recognition site is ggtgcc. The enzyme cleavage sites are used to distinguish between *Pomacea canaliculata* and *Pomacea cryptica*.

2. A PCR-RFLP rapid identification method of the Tarenia saginata and Tarenia cryptobolus mitochondrial COI gene, characterized in that, The identification method includes the following steps: 1) Extract genomic DNA from the sample to be tested, dilute it, and use it as template DNA; 2) Perform PCR amplification using template DNA to obtain PCR amplification products; 3) Based on the restriction site described in claim 1, the restriction endonuclease BshNI is used to perform an isothermal restriction digestion reaction on the PCR amplification product; wherein, the recognition site of the restriction endonuclease BshNI is ggtgcc. 4) The obtained enzyme digestion products are detected by gel electrophoresis to determine the type of sample to be tested; In the gel electrophoresis results of the enzyme digestion products, the sample tested when two bands were generated was *Pomacea canaliculata*, and the sample tested when one band was generated was *Pomacea cryptica*.

3. The rapid PCR-RFLP method for identifying the mitochondrial COI gene of *Pomacea canaliculata* and *Pomacea cryptica* according to claim 2, characterized in that, The enzyme digestion reaction system includes PCR amplification products, 10× enzyme digestion buffer, restriction endonuclease BshNI, and ddH2O.

4. The rapid PCR-RFLP method for identifying the mitochondrial COI gene of *Pomacea canaliculata* and *Pomacea cryptica* according to claim 2 or 3, characterized in that... The enzyme digestion reaction system included 10 μL of PCR amplification product, 2 μL of 10× enzyme digestion buffer, 1 μL of restriction endonuclease BshNI, and 7 μL of ddH2O.

5. The rapid PCR-RFLP method for identifying the mitochondrial COI gene of *Pomacea canaliculata* and *Pomacea cryptica* according to claim 2 or 3, characterized in that, The enzyme digestion reaction was carried out at a constant temperature of 65℃ for 2 hours.

6. The rapid PCR-RFLP method for identifying the mitochondrial COI gene of *Pomacea canaliculata* and *Pomacea cryptica* according to claim 2 or 3, characterized in that, The PCR amplification system includes template DNA, upstream and downstream primers, 10×Taq buffer, dNTPs, MgCl2, Taq enzyme, and ddH2O; The upstream and downstream primers are as follows: Upstream primer LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3'; Downstream primer HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'.

7. The rapid PCR-RFLP method for identifying the mitochondrial COI gene of *Pomacea canaliculata* and *Pomacea cryptica* according to claim 2 or 3, characterized in that... The PCR amplification system includes 1 μL template DNA, 1 μL each of 10 μmol / L upstream and downstream primers, 2.5 μL 10×Taq buffer, 2 μL 2.5 mmol / L dNTPs, 1 μL 25 mmol / L MgCl2, 0.25 μL 5 U / μL Taq enzyme, and ddH2O to a final volume of 25 μL. The upstream and downstream primers are as follows: Upstream primer LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3'; Downstream primer HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'.

8. The rapid PCR-RFLP method for identifying the mitochondrial COI gene of *Pomacea canaliculata* and *Pomacea cryptica* according to claim 2 or 3, characterized in that... The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ for 30 s, 50℃ for 30 s, 72℃ for 50 s, 35 cycles; 72℃ final extension for 10 min; and storage at 4℃.

9. The rapid PCR-RFLP method for identifying the mitochondrial COI gene of *Pomacea canaliculata* and *Pomacea cryptica* according to claim 2 or 3, characterized in that, Gel electrophoresis was performed using 1.5% agarose gel at 120V for 25-30 minutes.

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