Specific primers and rapid PCR method for identifying Philippine leech

Abstract

The present invention discloses specific primers for identifying the Philippine leech. The upstream primer L315 is: 5′-tgttggtacaggatgaacta-3′, and the downstream primer H566 is: 5′-gctgctgctaatactggta-3′. A rapid PCR method for identifying the Philippine leech is provided. The PCR reaction system comprises: 12.5 μL of Takara Premix Ex Taq, 1 μL each of the upstream primer L315 and the downstream primer H566, 1 μL of a DNA template, and sterile double-distilled water added to 25 μL. The PCR amplification procedure is: 94°C pre-denaturation for 1 minute, 94°C denaturation for 30 seconds, 52°C annealing for 30 seconds, for 28 cycles. The present invention addresses the shortcomings of existing PCR technology in the identification of Philippine leeches. Based on the SNP sites with stable differences in the CO1 sequence between Philippine leeches and other species, specific primers for Philippine leeches are designed, and a rapid PCR method is used to establish an identification method for Philippine leeches to distinguish them from other leeches.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology and more specifically relates to a specific primer and a rapid PCR method for identifying Hirudo philadelphica. Background Art

[0002] The Philippine leech (Poecilobdella manillensis Lesson), commonly known as the golden-edged leech, belongs to the genus Poecilobus, family Hirudinidae, order Arrhinidae, class Hirudinales, family Hirudinidae. It is widely distributed in Guangxi, Guangdong, and Fujian provinces of my country and is a specialty medicinal animal resource of Guangxi. It is included in the second volume of the 2011 edition of the "Quality Standards for Zhuang Medicines of Guangxi Zhuang Autonomous Region." Poecilobus contains a variety of active ingredients, including hirudin, phenanthellidone, plasmin, hyaluronidase, and platelet aggregation inhibitors, exhibiting anticoagulant, antithrombotic, antitumor, antigout, and lipid-lowering effects. Its anticoagulant activity is 3.6 times that of Hirudin japonicus and 13.2 times that of Hirudin spp. (Horticoides spp.) , respectively, demonstrating that Poecilobus has superior anticoagulant properties compared to other leech species. Furthermore, Poecilobus anticoagulant extracts contain a variety of proteins with potential medicinal properties, offering excellent nutritional and health benefits.

[0003] Hirudo philadelphica has high medicinal value and potential for cultivation, primarily through artificial breeding. However, adulteration, mixed species, and processing are common issues in the market. Traditional identification methods, such as phenotypic identification, require a high level of expertise and experience. Using only thin-layer chromatography (TLC) methods from pharmacopoeias presents limitations, particularly for traditional Chinese medicines containing Hirudo philadelphica, where the characteristic traits of the leech disappear, making traditional identification methods even less effective. Advances in molecular identification technology have provided new approaches for the identification of animal-derived traditional Chinese medicines. However, traditional DNA molecular identification methods are long and cumbersome, requiring electrophoresis, imaging, and sequencing after the PCR reaction, taking a significant time, typically 3–4 days. This significantly hinders their further application. Rapid PCR, a further development of specific PCR technology, combines rapid DNA extraction, rapid PCR amplification, and fluorescence detection to significantly shorten PCR reaction times and improve efficiency. At present, rapid PCR methods have been successfully applied to the authenticity identification and quality control of traditional Chinese medicines such as gecko, yam, toad oil, and Agkistrodon acutus, but there have been no reports on the identification of Philippine leech. Summary of the Invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] One purpose of the present invention is to provide a specific primer for identifying Hirudo philadelphica, which can provide a relatively fast, simple and effective Hirudo philadelphica identification method, provide technical support for the on-site application of Hirudo philadelphica molecular identification, and ensure the safety and efficacy of Hirudo philadelphica clinical medication.

[0006] In order to achieve these purposes and other advantages of the present invention, a specific primer for identifying Philippine leech is provided, wherein the upstream primer L315 is: 5′-tgttggtacaggatgaacta-3′, and the downstream primer H566 is: 5′-gctgctgctaatactggta-3′.

[0007] Preferably, the design of specific primers comprises the following steps:

[0008] 1) The COI sequences of Hirudo philippinosus, Hirudo japonica, Hirudo stylosus, and Hirudo tianmushanensis were downloaded from the NCBI database. The downloaded sequences were aligned using BioEdit software to identify variant sites with stable differences. Primers were designed in regions where Hirudo philippinosus differed significantly from Hirudo japonica, Hirudo stylosus, and Hirudo tianmushanensis.

[0009] 2) Specific primers were designed using the primer design software Premier Primer 5.0. The parameters were adjusted so that the 3' end of the primer was located in the difference region between Hirudo philippinosus and Hirudo japonica, Hirudo styracidae, and Hirudo tianmushanensis, with a Tm value of 55-65°C and an expected amplification length of 251 bp. The specific primers were obtained.

[0010] A PCR method for identifying Hirudo philadelphica comprises the upstream primer L315 and the downstream primer H566.

[0011] Preferably, the PCR reaction system is: Takara Premix Ex taq 12.5 μL, upstream primer L315, downstream primer H566 1 μL each, and the final primer concentration is 0.4 μM, DNA template 1 μL, and sterile double-distilled water is added to 25 μL;

[0012] The PCR amplification program was as follows: pre-denaturation at 94°C for 1 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, for 28 cycles.

[0013] Preferably, SYBR Green I is added to the PCR amplification product and fluorescence is detected at an ultraviolet wavelength of 365 nm. The product that produces strong green fluorescence is Hirudo philadelphica.

[0014] The present invention has at least the following beneficial effects:

[0015] First, the present invention addresses the shortcomings of existing PCR technology in the identification of Philippine leeches. Based on the SNP sites with stable differences in the CO1 sequence between Philippine leeches and other species, specific primers for Philippine leeches are designed, and a rapid PCR method is used to establish an identification method to distinguish Philippine leeches from other leeches.

[0016] Second, the present invention creatively combines rapid PCR technology to identify Philippine leech and uses alkaline lysis method to extract DNA, which is simpler and faster to operate than general extraction methods. The optimized PCR reaction parameters are also less time-consuming than general molecular identification methods.

[0017] Third, the present invention introduces a fluorescent dye to detect amplified products, enabling rapid identification. Samples can be identified based solely on fluorescence detection under ultraviolet light, eliminating the need for sequencing or other methods. This method is also suitable for rapid identification of large quantities of samples, significantly improving identification efficiency and addressing the issue of adulteration in the leech market. This is of great significance for ensuring the safety and efficacy of leech clinical use and regulating the leech market.

[0018] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The electrophoresis results of the PCR amplification products using the CO1 universal primer are shown in Figure 1. M is a D2000 DNA marker; W is a blank control; 1-10 are fresh Hirudo philippinosus; 11-13 are medicinal Hirudo philippinosus; 14-19 are Hirudo styracidae (medicinal material); 20-28 are Hirudo styracidae (fresh material); 29-37 are Hirudo japonica (fresh material); 38-43 are Hirudo tianmushanensis (fresh material);

[0020] Figure 2 The electrophoresis results of the PCR amplification products with specific primers are shown in Figure 2. M is D2000 DNA Marker; W is blank control; 1-10 are fresh Hirudo philippinosus; 11-13 are Hirudo philippinosus (medicinal material); 14-19 are Hirudo serrata (medicinal material); 20-28 are Hirudo serrata (fresh material); 29-37 are Hirudo japonica (fresh material); 38-43 are Hirudo tianmushanensis (fresh material);

[0021] Figure 3 This is the fluorescence detection result of rapid PCR amplification products. DETAILED DESCRIPTION

[0022] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0023] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0024] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0025] Example 1. Genomic DNA extraction and detection

[0026] 1. Solution Preparation

[0027] Solution A: Use a graduated cylinder to measure 97 mL of sterile water into a beaker, then use a pipette to draw 1 mL of Triton 100 (purity 98%) and stir to dissolve.

[0028] Solution B: Place 0.8g of solid sodium hydroxide in a small beaker and add 20mL of sterile water. Weigh 7.88g of solid Tris-HCl and place it in another small beaker. Initially add approximately 40mL of sterile water. Subsequently, add appropriate amounts of sodium hydroxide solution and sterile water in small amounts until the total volume reaches 50mL and the pH is 8. This yields 50mL of a 1M Tris-HCl solution (pH 8). Transfer 10mL of this solution to a glass bottle and dilute with 90mL of sterile water using a graduated cylinder to a 0.1M Tris-HCl solution (pH 8). Set aside.

[0029] Reagent C: Take 0.5 g of sodium hydroxide and 0.25 g of polyvinyl pyrrolidone respectively into a beaker, dissolve them in Solution A (25 mL) to prepare DNA extraction buffer, shake gently and set aside after complete dissolution.

[0030] 2. Extraction of Total Genomic DNA

[0031] Wipe the surface of the sample (sources listed in Table 1) with 75% alcohol and allow to dry. Take approximately 30 mg of fresh muscle tissue and mince it with surgical scissors. Wash with sterile water. Cut the medicinal herbs into small pieces. Place them into numbered 2 mL tubes. Sterilize the magnetic beads with 75% alcohol. Add sterilized, dried magnetic beads to each numbered tube and grind using a DNA extraction grinder to obtain the ground sample.

[0032] When using, add 200 μL of DNA extraction buffer to the ground sample and vortex to mix thoroughly; add 800 μL of solution B, vortex to mix thoroughly, and centrifuge at 12000 rpm for 1 min; take 500 μL of supernatant into a new 2 mL centrifuge tube, add 500 μL of solution B respectively, and vortex to mix thoroughly; store at -20℃ for later use.

[0033] Table 1 Sample sources

[0034]

[0035] 3. PCR amplification of target gene

[0036] The extracted DNA samples were amplified by PCR using CO1 universal primers (LCO1490: 5′-ggtcaacaaatcataaagatattgg-3′, HCO2198: 5′-taaacttcagggtgaccaaaaaatca-3′). The amplification conditions are shown in Table 2.

[0037] Table 2 Primers and PCR reaction conditions

[0038]

[0039] 4. PCR amplification product detection

[0040] Prepare 1% agarose gel by weighing 1g agarose into a conical flask, adding 100ml 1×TAE buffer solution and heating in a microwave oven for 3min. Pipette 1μL nucleic acid dye, add it to the dissolved gel solution, mix thoroughly and pour it into a gel plate. After 30min, the gel will form and be placed on the electrophoresis instrument. Add 1×TAE running buffer to soak the agarose gel.

[0041] Mix 3 μL of amplified DNA sample with 2 μL of 6× loading buffer. Then, apply 5 μL of the sample to each well. Also, apply 5 μL of D2000 DNA Marker and a blank control to each side of the sample. After sample loading, run the assay at 135 V for 30–40 minutes.

[0042] After electrophoresis, take out the gel and place it on the SYNGENE gel imaging system for observation and photography to test the quality of the extracted DNA. Figure 1 The results of agarose gel electrophoresis showed that the extracted DNA samples were able to amplify the target fragment and obtain a DNA band of about 700 bp, indicating that the DNA extracted from the samples using the alkaline lysis method was effective and could meet the needs of subsequent experimental research.

[0043] Example 2: Design and synthesis of specific primers

[0044] 1. Sequence Alignment

[0045] The COI sequences of Hirudo philippinosus, Hirudo japonica, Hirudo serrata, and Hirudo tianmushanensis were downloaded from the NCBI database (National Center for Biotechnology Information). The downloaded sequences were aligned using BioEdit software to identify variant sites with stable differences. Primers were designed in regions where Hirudo philippinosus differed significantly from other leech species.

[0046] 2. Primer Design

[0047] Specific primers were designed using the primer design software Premier Primer 5.0. Parameters were adjusted to ensure that the 3' end of the primer was located in a region that differs between the Philippine leech and other leech species, with a Tm of 55-65°C and an expected amplicon length of 251 bp. The final primers (L315: 5'-tgttggtacaggatgaacta-3', H566: 5'-gctgctgctaatactggta-3') were selected and synthesized by Nanning Genesis Biotechnology Co., Ltd.

[0048] Example 3, Optimization of PCR expansion conditions

[0049] Using genomic DNA from Hirudo philadelphica and other leech samples as templates, we optimized the specific identification primers L315 and H566 for rapid PCR amplification. We investigated the effects of annealing temperature, cycle number, annealing time, final primer concentration, template volume, different DNA enzyme types, and different PCR amplification instrument models on PCR reaction stability. Specific influencing factors are listed in Table 3.

[0050] Table 3 PCR reaction factor investigation table

[0051]

[0052] 1. Annealing temperature

[0053] Several key factors influence specific rapid PCR reactions. First, annealing temperatures were tested at 45°C, 52°C, 58°C, and 65°C. Results showed that at 45°C, the target band appeared in Hirudo philadelphica samples, while no other leech species showed it. However, the overall bands were somewhat faint. At 52°C, Hirudo philadelphica samples showed a clear, bright target band at approximately 251 bp, while no target band was detected in other leech samples. At 58°C, Hirudo philadelphica samples showed a bright target band, but one sample lacked it, and no other leech samples showed it. At 65°C, no target band was observed in Hirudo philadelphica samples or other leech samples. Fluorescence detection results were consistent with the electrophoresis results. It can be seen that among the four annealing temperatures, only 45°C and 52°C can completely amplify the target band, and the brightness of the target band at 52°C is stronger than that at 45°C. Therefore, the final annealing temperature selected is 52°C.

[0054] 2. Number of cycles

[0055] The specific rapid PCR reaction conditions were tested using 20, 28, 31, and 35 cycle times. The results showed that when the cycle number was set to 20, all leech samples showed the target band, but it was relatively light and the fluorescence was not obvious. No bands were observed in other leech samples. When the cycle number was set to 28, all leech samples showed bright target bands with obvious fluorescence, while no bands were observed in other leech samples. When the cycle number was set to 31 and 35, leech samples showed bright target bands, but occasionally, the bands were relatively light. The fluorescence detection results were consistent with the electrophoresis results. As can be seen, although the target bands appeared in all leech samples at all four cycle times, the PCR reaction time increased with each cycle number. The four cycle times (20, 28, 31, and 35) took 43, 55, 65, and 74 minutes, respectively. Therefore, considering the PCR amplification efficiency, the final number of cycles was 28.

[0056] 3. Annealing time

[0057] Annealing times were set to 5s, 10s, 20s, and 30s, respectively. The results showed that when the annealing time was 5s, the target bands of the Philippine leech samples were generally faint, with one sample lacking the target band, while all other leech samples had no target bands. When the annealing time was 10s, the target bands of the Philippine leech samples were also faint, with one sample lacking the target band and three samples having extremely faint bands, while all other leech samples had no target bands. When the annealing time was 20s, the target bands of the Philippine leech samples were bright, but three samples had extremely faint bands, while all other leech samples had no target bands. When the annealing time was 30s, all Philippine leech samples had bright target bands with obvious fluorescence, while all other leech samples had no bands. Therefore, the final annealing time was 30s.

[0058] 4. Final Primer Concentration

[0059] Final primer concentrations of 0.1 μM, 0.2 μM, 0.4 μM, and 0.6 μM were tested. Results showed that some Hirudo philadelphica samples lacked the target band at final primer concentrations of 0.1 μM, 0.2 μM, and 0.6 μM. However, at a final primer concentration of 0.4 μM, bright target bands were detected in all Hirudo philadelphica samples, while no bands were observed in other leech species. Fluorescence detection results were consistent with the electrophoresis results, with the most pronounced fluorescence at a concentration of 0.4 μM. Therefore, a final primer concentration of 0.4 μM was selected for the rapid PCR identification of Hirudo philadelphica.

[0060] 5. Template volume

[0061] Template volumes of 0.5 μL, 1 μL, 3 μL, and 6 μL were tested. The results showed that the target bands in the Philippine leech samples were generally faint when the template volumes were 0.5 μL, 3 μL, and 6 μL, respectively, while no target bands were observed in the other leech samples. At a template volume of 3 μL, one Philippine leech sample showed a band, while at a template volume of 6 μL, no bands were observed in six Philippine leech samples. Only at a template volume of 1 μL did all Philippine leech samples show a bright target band, while no bands were observed in the other leech samples. Fluorescence detection results were consistent with the electrophoresis results. Therefore, a template volume of 1 μL was ultimately selected for the Philippine leech rapid PCR identification method.

[0062] 6. Types of DNA enzymes

[0063] Takara Taq, Takara Ex Taq, and Tiangen Taq were tested. Agarose gel electrophoresis results showed that when Tiangen Taq was used, only one of the leech samples produced the target band; no other samples of the leech or other leeches showed the target band. When Takara Taq was used, some of the leech samples failed to amplify the target band. When Takara Ex Taq was used, bright target bands were detected in all leech samples, while no bands were observed in other leech samples. Therefore, Takara Ex Taq was ultimately selected for the rapid PCR identification of leech samples.

[0064] 7. PCR Amplifier

[0065] The PCR amplifiers investigated included the Bio-rad T100 PCR instrument from the United States, the Biometra PCR instrument from Jena, Germany, and the LightCycler PCR instrument from Roche, Switzerland. The results of agarose gel electrophoresis showed that all three PCR amplifiers could amplify the target bands in the Philippine leech samples, and the differences were not significant. The target bands could not be amplified in other leech samples. The fluorescence detection results were consistent with the electrophoresis results, indicating that the PCR reaction conditions were stable in the amplification results of PCR amplifiers from different manufacturers and models.

[0066] Based on the above experimental results, the rapid PCR identification method for leech P. philippinarum was determined as follows: a 25 μL PCR amplification reaction system consisting of 12.5 μL Takara Premix Ex Taq, 1 μL each of upstream and downstream primers (final primer concentration 0.4 μM), and 1 μL DNA template, brought to 25 μL with sterile double-distilled water. The PCR amplification procedure consisted of 28 cycles of pre-denaturation at 94°C for 1 minute, denaturation at 94°C for 30 seconds, and annealing at 52°C for 30 seconds. PCR amplification products were detected by 1% agarose gel electrophoresis. All leech samples showed a clear, specific band around 251 bp, while other leech samples showed no band. Addition of 2 μL of 100× SYBR Green I dye to the amplified product revealed bright green fluorescence in leech P. philippinarum samples, while other leech samples showed no fluorescence.

[0067] Example 4: PCR Identification of Hirudophila

[0068] The DNA was extracted by alkaline lysis method, and the optimized PCR reaction parameters were selected to amplify the samples of Hirudo philippinarum and its mixed products. The results of agarose gel electrophoresis showed that the samples of Hirudo philippinarum could be effectively amplified, with a clear band at 251 bp, such as Figure 2As shown; 2 μL 100×SYBR Green I was added to the PCR amplification product and fluorescence was detected at 365 nm ultraviolet wavelength. The Philippine leech samples all produced strong green fluorescence, while the other samples had no fluorescence. The results are shown in Figure 3 . The results were consistent with the gel electrophoresis results, and the identification results were accurate.

[0069] The rapid PCR identification method of the present invention takes less than 10 minutes to extract genomic DNA and about 50 minutes for PCR amplification. The entire molecular identification operation of the Philippine leech can be completed within 1 hour.

[0070] Using conventional PCR identification methods, such as the one provided in application number CN201810824002.0, genomic DNA extraction requires at least 3 hours, PCR amplification requires at least 2.5 hours, and agarose gel electrophoresis requires 30 minutes. The entire molecular identification process for Hirudo philadelphica takes at least 6 hours. This significantly improves the identification efficiency of the present method.

[0071] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A specific primer for identifying Hirudo philippinosus, characterized in that: The upstream primer L315 is: 5′-tgttggtacaggatgaacta-3′, and the downstream primer H566 is: 5′-gctgctgctaatactggta-3′.

2. A rapid PCR method for identifying Hirudo philippinarum, characterized in that: The method comprises performing PCR using the upstream primer L315 and the downstream primer H566 described in claim 1.

3. The rapid PCR method for identifying Hirudo philadelphica according to claim 2, characterized in that: The PCR reaction system was as follows: 12.5 μL of Takara Premix Ex taq, 1 μL each of upstream primer L315 and downstream primer H566, with the final primer concentration at 0.4 μM, 1 μL of DNA template, and sterile double-distilled water to 25 μL. The PCR amplification program was as follows: pre-denaturation at 94°C for 1 min, denaturation at 94°C for 30 s, and annealing at 52°C for 30 s, for 28 cycles.

4. The rapid PCR method for identifying Hirudo philadelphica according to claim 2, wherein: SYBR Green I was added to the PCR amplification product and fluorescence was detected at an ultraviolet wavelength of 365 nm. The amplification product that produced green fluorescence was Hirudo philadelphica.

Images