A lamp primer set, kit, method and application for detecting helicid snails

Abstract

The application belongs to the technical field of biological detection and identification, and particularly relates to a LAMP primer set, kit, method and application for detecting and identifying Bradybaena fruticum. The LAMP primer set for detecting Bradybaena fruticum comprises an outer primer set and an inner primer set. The outer primer set comprises HP3F3 shown in SEQ ID NO:1 and HP3B3 shown in SEQ ID NO:2, and the inner primer set comprises HP3FIP shown in SEQ ID NO:3 and HP3BIP shown in SEQ ID NO:4. The LAMP primer set, kit and method are high in specificity and sensitivity, can provide rich identification basis at the gene level, and can effectively distinguish interspecific differences.

Description

Technical Field

[0001] This invention belongs to the field of biological detection and identification technology, specifically relating to a LAMP primer set, reagent kit, method, and application for detecting and identifying the giant scabbard snail. Background Technology

[0002] The giant cap snail (Helix pomatia) belongs to the phylum Mollusca, class Gastropoda, subclass Pulmonata, order Stylommatopgora, family Helicidae, and genus Helix. This snail not only directly damages fruits, vegetables, and other crops, but wild snails can also transmit zoonotic parasitic diseases, making it a significant potential threat to agriculture, natural ecosystems, human health, and trade. Originating in Europe, the giant cap snail has now invaded countries such as Canada, the United States, and Australia, but is not yet found in my country. In recent years, this snail has been frequently intercepted at ports of entry in my country, including Beijing. Risk assessments indicate a very high risk of its introduction into my country, necessitating strengthened quarantine measures to prevent its spread.

[0003] Currently, the detection and identification of giant snails, both domestically and internationally, mainly relies on shell morphology, reproductive system anatomy, and conventional PCR amplification and sequencing comparison. However, key identification characteristics of snail shells are often variable due to factors such as geographical environment and climate, while reproductive system anatomy requires specialized personnel in a professional laboratory with specialized equipment, which is difficult for frontline port personnel and grassroots laboratories. Conventional PCR (polymerase chain reaction) amplification and sequencing processes take at least two days to complete, and sending sequencing out for testing creates a certain economic burden. Therefore, existing methods not only rely on professional identification experience and sophisticated equipment but are also time-consuming and labor-intensive, making them difficult to implement at quarantine sites and grassroots laboratories.

[0004] Loop-mediated isothermal amplification (LAMP) is a rapidly developing molecular biology technique, playing a crucial role, particularly in biological detection. It has been widely applied to viruses, bacteria, and fungi, but its application in snail detection and identification remains a gap in the field. To date, no research has been found on LAMP primer sets, kits, or methods for snail detection and identification. Summary of the Invention

[0005] The technical problem to be solved by this invention is that the identification of the giant slug snail is difficult, time-consuming and labor-intensive, and cannot meet the needs of rapid customs clearance at ports and prevention and control in grassroots laboratories.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: a LAMP primer set for detecting the giant slug snail, comprising an outer primer set and an inner primer set. The outer primer set includes the forward outer primer HP3F3 shown in SEQ ID NO:1 and the reverse outer primer HP3B3 shown in SEQ ID NO:2. The inner primer set includes the forward inner primer HP3FIP shown in SEQ ID NO:3 and the reverse inner primer HP3BIP shown in SEQ ID NO:4, as shown in Table 1.

[0007] Table 1

[0008] Another technical solution adopted in this invention is: a kit for detecting *Gnaphalium affine*, comprising reaction mixture I, reaction mixture II, a positive control, a negative control, a fluorescent dye, and ddH2O. The reaction mixture II comprises an outer primer set and an inner primer set. The outer primer set comprises HP3F3 shown in SEQ ID NO:1 and HP3B3 shown in SEQ ID NO:2, and the inner primer set comprises HP3FIP shown in SEQ ID NO:3 and HP3BIP shown in SEQ ID NO:4, as shown in Table 1.

[0009] Another technical solution adopted in this invention is: a method for detecting the captive snail, comprising the following steps: S1: Extract DNA from the sample; S2: The samples were subjected to LAMP detection using the above-mentioned kit for detecting scabbard snails.

[0010] Another technical solution adopted in this invention is the application of the above-mentioned reagent kit for detecting capped giant snails in the detection of capped giant snails.

[0011] The beneficial effects of this invention are as follows: Addressing the shortcomings of traditional morphological and conventional PCR identification techniques, this invention provides a set of highly specific and sensitive LAMP primers and a detection kit. Detection using this kit is unaffected by individual morphology, size, or integrity, avoiding the limitations of morphological identification. It provides rich identification evidence directly at the gene level, effectively distinguishing interspecies differences. In practical work, anyone with basic molecular manipulation skills can use this kit and method for detection and identification; the operation is simple and reliable. The LAMP kit and method established in this invention provide a new technical means for the accurate detection and identification of *Gnaphalium affine*, filling a technological gap in the industry both domestically and internationally. Attached Figure Description

[0012] Figure 1The LAMP detection results of Example 3 of the present invention are shown in the figure. In the figure, a is the turbidimetric amplification curve, b is the reaction tube color diagram, 1 is the giant slug snail, 2 is the milky auricularia snail, 3 is the worm snail Yin's snail, 4 is the giant svenhoe snail, 5 is the wrinkled snail, 6 is the pizza tea snail, 7 is the forest onion snail, 8 is the giant slug snail, 9 is the garden onion snail, 10 is the giant African snail, and CK is the blank control. Figure 2 The following are the LAMP detection results of this invention at different concentrations of *Cape Magna globosa* DNA, where a is the turbidimetric amplification curve, b is the reaction tube color chart, and 1 represents 10. 2 ng / μL, 2 is 10 1 ng / μL, 3 is 10 0 ng / μL, 4 is 10 -1 ng / μL, 5 is 10 - 2 ng / μL, 6 is 10 -3 ng / μL, CK is the blank control; Figure 3 The LAMP detection results for this invention using different primer sets are shown in Figure a, where a is the turbidimetric amplification curve and b is the color chart of the reaction tube. Figure 4 The LAMP detection results for different amplification temperatures in this invention are shown, where a is the turbidimetric amplification curve and b is the reaction tube color chart. Figure 5 The LAMP detection results of *Gastropoda melanogaster* from different countries of origin are shown in the figure. In the figure, a is the turbidimetric amplification curve, b is the reaction tube color graph, 1 is from Romania, 2 is from Slovakia, 3 is from Germany, 4 is from the Netherlands, 5 is the negative control, and CK is the blank control. Detailed Implementation

[0013] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0014] A LAMP primer set for detecting the giant slug snail includes an outer primer set and an inner primer set. The outer primer set includes HP3F3 shown in SEQ ID NO:1 and HP3B3 shown in SEQ ID NO:2, and the inner primer set includes HP3FIP shown in SEQ ID NO:3 and HP3BIP shown in SEQ ID NO:4, as shown in Table 1.

[0015] As can be seen from the above description, the beneficial effects of the present invention are as follows: the LAMP primer set of the present invention has high specificity and high sensitivity. The four primers can perform six-fold control on the target species. There is no non-specific amplification for similar species. It can be used to quickly detect and identify giant stag snails to prevent the invasion of harmful organisms, maintain national biosecurity, and protect agricultural and forestry production and human and animal health.

[0016] This invention designs LAMP primers for the ITS2 gene of the giant slug snail and uses them for the first time in LAMP detection of the snail. However, without technical references for related species identification methods, designing and screening these specific primers presents significant challenges. The selected specific primers require verification of important parameters such as primer complementarity, complementarity between primers, GC content (the ratio of cytosine and guanine bases in the DNA or RNA sequence), and primer end stability. Care must also be taken to avoid aerosol contamination and false positives caused by primer dimers. Most importantly, the verification experiment must be scientifically designed based on species relationships to ensure the uniqueness of the primers to the giant slug snail.

[0017] Another technical solution adopted in this invention is: a kit for detecting *Gnaphalium affine*, comprising reaction mixture I, reaction mixture II, a positive control, a negative control, a fluorescent dye, and ddH2O. Reaction mixture II comprises an outer primer set and an inner primer set; the outer primer set comprises HP3F3 shown in SEQ ID NO:1 and HP3B3 shown in SEQ ID NO:2, and the inner primer set comprises HP3FIP shown in SEQ ID NO:3 and HP3BIP shown in SEQ ID NO:4.

[0018] As described above, the kit of the present invention has high sensitivity, and the detection concentration for *Gastropoda scaphina* can be as low as 10. -2 ng / μL. The kit provided by this invention has high specificity, showing a typical amplification curve only for *Cape-covered snail*, while no amplification curves were observed for other species.

[0019] Furthermore, reaction mixture I includes DNA polymerase.

[0020] Furthermore, reaction mixture I comprises 2×Reaction Mix.

[0021] Furthermore, the 2×Reaction Mix includes dNTPs (deoxyribonucleoside triphosphates), Mg 2+ and buffer Furthermore, the molar ratio of HP3F3, HP3B3, HP3FIP and HP3BIP in reaction mixture II is 1:1:8:8.

[0022] Furthermore, the positive control was the whole genome DNA of the giant cap snail.

[0023] Furthermore, the negative control was DNA from uncapped giant snails.

[0024] Another technical solution adopted in this invention is: a method for detecting the captive snail, comprising the following steps: S1: Extract DNA from the sample; S2: The samples were subjected to LAMP detection using the above-mentioned kit for detecting scabbard snails.

[0025] Furthermore, after the reaction, the results were determined based on the amplification curve on the turbidimeter and the colorimetric analysis of the reaction tube. The determination method is as follows: the positive control shows an S-shaped amplification curve on the turbidimeter and the reaction tube is green; the blank control and negative control both show a horizontal straight line on the turbidimeter and the reaction tube is orange-yellow. When the sample to be tested shows an S-shaped amplification curve on the turbidimeter or the reaction tube is green, the sample is determined to be a capped giant snail. When the sample to be tested shows a horizontal straight line on the turbidimeter or the reaction tube is orange-yellow, it is determined to be an uncapped giant snail or the DNA template concentration is too low.

[0026] As described above, the detection method of this invention has high sensitivity, strong specificity, short processing time, simple operation, accurate result determination, and no need for PCR post-processing, effectively avoiding false positives and cross-contamination. After the reaction, the amplification curve is determined by a turbidimeter or by direct observation of the reaction tube color, providing a clear and visual result. It can be used for rapid and visual detection and identification of *Gastropoda squarrosa* and its similar species: *Auricularia auricula-judae*, *Synthia svenhoei*, *Synthia squarrosa*, *Tea pissa*, *Onion snail*, *Gastropoda squarrosa*, *Gastropoda squarrosa*, and *Gastropoda squarrosa*.

[0027] Furthermore, the amplification temperature for LAMP detection is 58~64℃.

[0028] As described above, *Gastropoda spp.* showed positive amplification at temperatures ranging from 58 to 64°C.

[0029] Furthermore, the reaction procedure for LAMP detection is as follows: first, amplification is carried out at an isothermal temperature of 58-64℃, and then inactivation is carried out at 80℃.

[0030] Furthermore, the reaction procedure for LAMP detection is as follows: first, amplify at 58~64℃ for 60 min, and then inactivate at 80℃ for 5 min.

[0031] As described above, the detection method of this invention does not require complex instruments or special reagents; only a turbidimeter or constant temperature heater is needed for reaction and detection, making it simple and convenient to operate. This invention can complete the detection and identification of *Gastropoda fasciata* in approximately 60 minutes, which is fast.

[0032] Another technical solution adopted in this invention is the application of the above-mentioned reagent kit for detecting capped giant snails in the detection of capped giant snails.

[0033] As described above, when traditional taxonomic methods are insufficient to identify *Scaphina spp.*, the method of this invention uses molecular biology techniques to compensate, overcoming professional barriers and enabling rapid, accurate, and efficient detection and identification of *Scaphina spp.* from different countries of origin.

[0034] Embodiment 1 of the present invention is: a LAMP primer set for detecting the giant slug snail. The LAMP primer set consists of an outer primer set and an inner primer set. The outer primer set includes HP3F3 shown in SEQ ID NO:1 and HP3B3 shown in SEQ ID NO:2. The inner primer set includes HP3FIP shown in SEQ ID NO:3 and HP3BIP shown in SEQ ID NO:4, as shown in Table 1.

[0035] The fluorescent dyes used in the following examples were all from the Loopamp Fluorescence Visual Detection Kit (SLP221); the reaction mixture I (2×Reaction Mix and DNA polymerase) was all from the Loopamp DNA Amplification Kit (SLP204).

[0036] Embodiment 2 of the present invention is: a kit for detecting the giant slug snail, the components of which are shown in Table 2, and the specific sequences of the primers in Table 2 are shown in Table 1.

[0037] Table 2

[0038] The third embodiment of this invention is a method for detecting the captive snail, the specific steps of which are as follows: S1. The following snails were selected as test snails: Giant Cap Snail, Mammary Ear Snail, Venus yin's Snail, Giant Svenhoe's Snail, Scaly Snail, Pizza Snail, Forest Onion Snail, Giant Wild Snail, Garden Onion Snail, and Giant African Snail. These test snails were intercepted or collected through quarantine at ports of entry nationwide. After morphological and molecular confirmation by the National Key Laboratory for Quarantine and Identification of Molluscs of the General Administration of Customs, they were stored at -20℃ for later use. Sterile water served as a blank control. DNA was extracted from the tested snails using the TIANGEN tissue genomic DNA extraction kit, following the instructions for use. The concentration of DNA was then measured using a NanoDrop micro-nucleic acid protein analyzer. If the DNA concentration was greater than 100 ng / μL, the DNA was diluted to 100 ng / μL with deionized water and stored at -20℃ for later use to obtain the DNA template.

[0039] S2. Perform LAMP amplification on the DNA template under isothermal conditions, with two replicates for each template; The 25 μL LAMP reaction system consisted of: 12.5 μL of 2×Reaction Mix, 1 μL each of external primers HP3F3 and HP3B3 (both 5 pmol / μL), 1 μL each of internal primers HP3FIP and HP3BIP (both 40 pmol / μL), 1 μL of DNA polymerase (8 U), 2 μL of DNA template (100 ng / μL), 1 μL of fluorescent dye (25 μmol / L), and deionized water to a final volume of 25 μL.

[0040] The LAMP amplification reaction conditions are: isothermal amplification at 62℃ for 60 min, inactivation at 80℃ for 5 min, and then the reaction is terminated.

[0041] S3. Judgment: After the reaction was completed, the results were determined based on the turbidimeter amplification curve and the colorimetric analysis of the reaction tubes. (See attached image) Figure 1 , Figure 1 Table a shows the turbidimetric amplification curve, and table b shows the color chart of the reaction tubes. 1 is *Gnaphalium affine*, 2 is *Auricularia amygdala*, 3 is *Sinonovacula yinni*, 4 is *Gnaphalium svenhoei*, 5 is *Sinonovacula scabra*, 6 is *Sinonovacula pisaca*, 7 is *Sinonovacula forestensis*, 8 is *Sinonovacula sanguisorba*, 9 is *Sinonovacula gardenensis*, and 10 is *Gnaphalium affine*. CK is the blank control. Figure 1 The results showed that the capped snail exhibited positive amplification, and the LAMP reaction tube solution changed from orange-yellow to green. The other nine snail species and the blank control showed no amplification, and their reaction tubes remained orange-yellow. This demonstrates that the LAMP detection method of this invention has good specificity.

[0042] Based on Example 3, the following tests were performed: (a) Sensitivity test: The DNA of *Spodoptera exigua* was diluted to a concentration of 10-fold using sterile water in a serial 10-fold dilution method. 2 ng / μL, 10 1 ng / μL, 10 0 ng / μL, 10 -1 ng / μL, 10 -2 ng / μL, 10 -3 Different concentration gradients of ng / μL were used, with 2μL of dilution solution as DNA template and sterile water as blank control. LAMP amplification reaction was carried out according to the LAMP detection system and procedure in Example 3. Each concentration was repeated twice, and the detection results were observed using a real-time turbidimeter and a fluorescent dye method, respectively. Test results are shown Figure 2 , Figure 2 In the diagram, a is the turbidimetric amplification curve, b is the reaction tube color chart, and 1 represents 10. 2 ng / μL, 2 is 10 1 ng / μL, 3 is 100 ng / μL, 4 is 10 -1 ng / μL, 5 is 10 -2 ng / μL, 6 is 10 -3 ng / μL, CK is the blank control. Figure 2 The results showed that when the DNA concentration of the giant slug snail was diluted to 10... -2 Positive amplification was observed at concentrations of ng / μL, and the corresponding LAMP reaction tubes turned green. The experimental results demonstrate that the LAMP detection method established in this invention has high sensitivity and can detect concentrations up to 10 ng / μL. -2 ng / μL.

[0043] (II) Primer screening experiment: First, the sequences of the target species and similar species were amplified using universal ITS2 primers. After confirming that the sequences were correct, sequence alignment was performed to identify interspecific variations and conserved regions within the species. LAMP primers for the ITS2 gene of *Gnaphalium affine* were designed using the online software PrimerExplorV5. The five sets of primer sequences designed are shown in Table 3. According to the LAMP detection system in Example 3, using the genomic DNA of *Gnaphalium affine* as a template and sterile water as a blank control (CK), LAMP amplification was performed. The LAMP amplification conditions were set as follows: isothermal amplification at 60℃ for 60 min, followed by inactivation at 80℃ for 5 min. Each set of primers was tested twice, and the detection results were observed using a real-time turbidimeter and a fluorescent dye method, respectively. Table 3

[0044] Test results are shown Figure 3 , Figure 3 Image a shows the turbidimetric amplification curve, and image b shows the color of the reaction tubes. Figure 3 As can be seen from diagram a, both replicates of the HP2, HP3, and HP4 primer sets showed obvious amplification curves, with the HP3 primer set undergoing the LAMP reaction first. (The reaction tubes were observed visually.) Figure 3 (b) The solutions in the reaction tubes containing the above three primer sets were all green, while the solutions in the other reaction tubes were orange-yellow, consistent with the results detected by the real-time turbidimeter. Therefore, the HP3 primer set was determined to be the optimal primer set for the LAMP detection system of capped giant snails.

[0045] (III) Optimization of reaction temperature.

[0046] Using the genomic DNA of the giant slug snail as a template and sterile water as a blank control (CK), the LAMP detection system of Example 3 was used to amplify the DNA at 56℃, 58℃, 60℃, 62℃, 64℃, and 66℃ for 60 min, and then inactivate the DNA at 80℃ for 5 min. The LAMP amplification reaction was repeated twice at each temperature, and the detection results were observed using a real-time turbidimeter and a fluorescent dye method. Test results are shown Figure 4 , Figure 4 Image a shows the turbidimetric amplification curve, and image b shows the color of the reaction tubes. Figure 4 It can be seen that the capped snail showed positive amplification at 58~64℃, with the earliest amplification occurring at 62℃, and the solutions in the reaction tubes at the corresponding temperatures all turned green. Therefore, the optimal reaction temperature was determined to be 62℃.

[0047] (iv) LAMP test of giant scabbard snails from different countries of origin Using genomic DNA from four different countries—Romania, Slovakia, Germany, and the Netherlands—as templates, sterile water as a blank control, and *Gastropoda spp.* as a negative control, LAMP amplification reactions were performed according to the LAMP detection system and procedure in Example 3. Each template was tested twice, and the detection results were observed using a real-time turbidimeter and a fluorescent dye method, respectively. Test results are shown Figure 5 , Figure 5 Table a shows the turbidimetric amplification curve, and table b shows the color chart of the reaction tubes. Tube 1 represents tubes from Romania; tube 2 from Slovakia; tube 3 from Germany; tube 4 from the Netherlands; tube 5 is the negative control; and CK is the blank control. Figure 5 The results showed that the capped giant snails from the four source countries all exhibited obvious amplification curves, and the corresponding LAMP reaction tubes were green, indicating that the LAMP detection method established in this invention can be applied to the detection of actual samples.

[0048] The fourth embodiment of this invention is the application of the reagent kit from the second embodiment in the detection of capped giant snails.

[0049] In summary, the LAMP primer set, kit, method, and application for detecting *Gastropoda occulta* provided by this invention have the following advantages: 1. High specificity: The four primers can perform six-fold control on the target species, and there is no non-specific amplification for similar species.

[0050] 2. High sensitivity: The detection concentration for *Gastropoda septemlobus* can reach as low as 10. -2 ng / μL.

[0051] 3. Visual and intuitive result determination: The primer set, reagent kit and method designed in this invention for detecting and identifying *Gastropoda stenoptera* have been tested and verified on nine representative snail species, including *Auricularia auricula-judae*, *Syntos svenhoei*, *Syntos svenhoei*, *Syntos svenhoei*, *Syntos svenhoei*, *Syntos svenhoei*, *Syntos sacchar ... and *Syntos sacchari*. After the reaction, the results are determined by the amplification curve of a turbidimeter or by direct observation of the color of the reaction tube, making the results visual and intuitive.

[0052] 4. Simple and fast operation: No complicated instruments or special reagents are required. Only a turbidimeter or constant temperature heater is needed for reaction and detection. The operation is simple and convenient. This invention can complete the detection and identification of the giant slug snail in about 60 minutes, which is fast.

[0053] 5. High Practicality: When traditional taxonomic methods are insufficient to identify *Scaphina scutellario*, this invention utilizes molecular biology techniques to overcome professional barriers, enabling rapid, accurate, and efficient detection and identification of *Scaphina scutellario*. Therefore, this method is highly practical and meets the needs for rapid and reliable detection and identification of *Scaphina scutellario*.

[0054] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A LAMP primer set for detecting the giant scabbard snail, characterized in that, It includes an outer primer set and an inner primer set, wherein the outer primer set includes HP3F3 shown in SEQ ID NO:1 and HP3B3 shown in SEQ ID NO:2, and the inner primer set includes HP3FIP shown in SEQ ID NO:3 and HP3BIP shown in SEQ ID NO:

4.

2. A reagent kit for detecting *Capetopus scabra*, characterized in that, The mixture includes reaction mixture I, reaction mixture II, a positive control, a negative control, a fluorescent dye, and ddH2O. Reaction mixture II includes an outer primer set and an inner primer set. The outer primer set includes HP3F3 shown in SEQ ID NO:1 and HP3B3 shown in SEQ ID NO:2, and the inner primer set includes HP3FIP shown in SEQ ID NO:3 and HP3BIP shown in SEQ ID NO:

4.

3. The reagent kit for detecting *Gastropoda fasciata* according to claim 2, characterized in that, The reaction mixture I includes DNA polymerase.

4. The reagent kit for detecting *Gastropoda fasciata* according to claim 2, characterized in that, The reaction mixture I comprises 2×Reaction Mix.

5. The reagent kit for detecting *Gastropoda fasciata* according to claim 2, characterized in that, The molar ratio of HP3F3, HP3B3, HP3FIP and HP3BIP in the reaction mixture II is 1:1:8:

8.

6. The reagent kit for detecting *Gastropoda fasciata* according to claim 2, characterized in that, The positive control was the whole genome DNA of the giant scabbard snail.

7. The reagent kit for detecting *Gastropoda fasciata* according to claim 2, characterized in that, The negative control was DNA from the uncapped giant snail.

8. A method for detecting the captive snail, characterized in that, Includes the following steps: S1: Extract DNA from the sample; S2: The sample was subjected to LAMP detection using the kit for detecting the captive snail as described in any one of claims 2-7.

9. The method for detecting the giant slug snail according to claim 8, characterized in that, The amplification temperature for LAMP detection is 58~64℃.

10. The application of the kit for detecting *Gastropoda fasciata* as described in any one of claims 2-7 in the detection of *Gastropoda fasciata*.

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