Method for detecting whitmania algam

Abstract

The invention aims to provide a method for detecting whitmania algam. The Latin name of the whitmania algam leech is Zeylanica argumensis, and the Latin name of the whitmania algam leech is Zeylanica argumensis; the method comprises the following steps: detecting the COI gene sequence of the algam tin orchid by using an enzymatic isothermal amplification (ERA) technology; the primer sequence of the ERA is as follows: ZE-PAGE-F1; the preparation method comprises the following steps: preparing a ZE-HPLC ( According to the direction from the 5'tail end to the 3 'tail end, the nucleotide sequence of the ZE-PAGE-F1 is a sequence number (ID): 1; the nucleotide sequence of the ZE-HPLC-R1 is shown as a sequence number (ID): 2. The method does not depend on a PCR instrument, has the advantages of being easy and convenient to operate, short in reaction time, high in sensitivity and the like, can detect the pathogenic nucleic acid with the minimum 1 * 10 < 1 > copies / microliter, and greatly improves the detection efficiency.

Description

Technical Field

[0001] The invention relates to a detection method for Arugam Ceylon leech, and belongs to the technical field of detection. Background Art

[0002] The Arugamensis leech (Zeylanicobdella arugamensis), belonging to the genus Zeylanicum (Annelida) and the phylum Annelida, is an obligate ectoparasite of marine fish. It has been reported to infect over 30 species of marine fish, including the leopard gill bass (Plectropomus leopardus), the cog-banded grouper (Epinephelus coioides), the saddle-banded grouper (E. lanceolatus), and the purple snapper (Lutjanus argentimaculatus), causing significant damage. Its simple life cycle, consisting of two stages, egg cocoon incubation and juvenile leech development, has led to its widespread prevalence in marine aquaculture environments.

[0003] The Arugam Ceylon leech parasitizes the host's fins and other surface tissues. Its attachment behavior can cause erosion, inflammation, and congestion of the host's skin, leading to severe damage to the epidermis. The parasite obtains nutrition by sucking blood until it matures, separates from the host, lays eggs in a cocoon, and dies. In addition, the Arugam Ceylon leech can serve as a vector for bacteria, viruses, fungi, and blood parasites, exacerbating outbreaks of fish diseases. It has been reported that the parasite can cause secondary infections, such as Vibrio alginolyticus infection, and the host usually dies within 3 days of infection. Therefore, the Arugam Ceylon leech is considered one of the high-risk external parasites that endanger many types of fish.

[0004] Although research on the Arugam leech has made some progress both domestically and internationally, primarily focusing on histopathology, taxonomic identification, host range, life history, and drug control, practical prevention and control still face numerous challenges. Currently, there is a lack of effective control measures for this parasite, hindering fundamental control of infection. Therefore, developing rapid and accurate detection technologies to enable early identification and dynamic monitoring of parasites is crucial for effective control. This not only provides a scientific basis for the timely implementation of control measures but also significantly reduces the incidence and spread of the disease, possessing important theoretical and practical significance.

[0005] Enzymatic Recombinase Amplification (ERA) is an efficient and simple constant-temperature nucleic acid amplification technique that has been widely used in the detection of aquatic pathogens. Compared with traditional PCR, ERA does not require extensive instrumentation and can achieve highly sensitive nucleic acid amplification at a constant temperature of 25°C to 45°C. Furthermore, ERA combined with lateral flow dipsticks (LFD) (ERA-LFD) allows for rapid visualization of results, making it suitable for on-site pathogen detection. ERA-LFD distinguishes positive from negative samples through immunochromatographic detection using a FAM (FITC)-biotin reporter gene. When the target pathogen is present, the gold-labeled anti-biotin antibody fully binds to the FAM-biotin, resulting in a signal on the detection strip; otherwise, the signal on the detection strip decreases.

[0006] While ERA technology has demonstrated significant value in pathogen detection for aquatic diseases, no research has been reported on the Arugam leech. The development of rapid ERA-LFD-based detection technology is expected to enable early diagnosis and dynamic monitoring of Arugam leech infection, providing technical support for disease prevention and control, and has significant research and application value. Summary of the Invention

[0007] The invention aims to provide a method for detecting the Arugam Ceylon leech.

[0008] To achieve the purpose of the present invention, the Latin name of the Arugam Ceylon leech is Zeylanicobdella arugamensis; the method comprises detecting the Arugam Ceylon COI gene sequence using enzymatic isothermal amplification technology ERA;

[0009] The primer sequences of the ERA are: ZE-PAGE-F1; ZE-HPLC-R1; in the direction from the 5' end to the 3' end, the nucleotide sequence of the ZE-PAGE-F1 is sequence number (ID): 1; the nucleotide sequence of the ZE-HPLC-R1 is sequence number (ID): 2.

[0010] In a specific embodiment, the ZE-PAGE-F1 primer is purified by polyacrylamide gel electrophoresis, and the ZE-HPLC-R1 primer is purified by high performance liquid chromatography.

[0011] In a specific embodiment, the length of the primer is 220 bp.

[0012] In one embodiment, the ERA is an ERA combined with a lateral flow chromatography test strip.

[0013] In a specific embodiment, the ERA probe is a ZE-HPLC-probe; in the direction from the 5' end to the 3' end, the nucleotide sequence of the ZE-HPLC-probe is sequence number (ID): 15; a FAM fluorescent group is set at the 5' end of sequence number (ID): 15, and a C3-Spacer blocking group is set at the 3' end, and a base located 30 bp downstream of the FAM fluorescent group and 15 bp upstream of the C3-Spacer blocking group is replaced with tetrahydrofuran.

[0014] In a specific embodiment, the probe is 47 bp in length.

[0015] In a specific embodiment, the primer for the ERA-binding lateral flow chromatography test strip is ZE-HPLC-R1-biotin, and the ZE-HPLC-R1-biotin is labeled with vitamin B7 at the 5' end of ZE-HPLC-R1.

[0016] In a specific embodiment, the reaction temperature of the ERA combined with the lateral flow chromatography test strip is 39° C. to 45° C.; and the reaction time is 5 to 30 minutes.

[0017] In a specific embodiment, the reaction temperature of the ERA combined with the lateral flow chromatography test strip is 41° C. and the reaction time is 10 min.

[0018] In one embodiment, the detection limit of the ERA is 1.6×10 1 copies / μL.

[0019] Beneficial effects:

[0020] The ERA-LFD method established in the present invention has the advantages of strong specificity, high sensitivity, and simple operation. It can quickly and accurately detect the pathogen of Arugam ceylon leech in tissues and culture environments, providing important technical support for the prevention and control of Arugam ceylon leech disease, specifically:

[0021] 1. The present invention does not require PCR instruments, is easy to operate, and can complete the detection within 10 to 20 minutes, greatly improving the detection efficiency.

[0022] 2. The method of the present invention can detect pathogen nucleic acids at concentrations as low as 1.6×101 copies / μL. Testing of a large number of fish tissue and water samples showed that the ERA-LFD method produced results consistent with those of the basic ERA method, while also demonstrating higher sensitivity than conventional PCR.

[0023] 3. The method of the present invention has no cross-reaction to common pathogens of marine fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Photos of marine fish and worms infected with Arugam Ceylon leech; Figure 1 Middle A: A leopard gill bass infected with the Arugam Ceylon leech, with visible worms (as indicated by the red arrows) and damaged dorsal fin skin; B: A greater amberjack infected with the Arugam Ceylon leech, with visible worms (as indicated by the red arrows); C: A photo of the Arugam Ceylon leech under an optical microscope, showing a pair of black triangular eyespots on the back of the front sucker and a long snout on the neck.

[0025] Figure 2 The results of primer screening for Arugam Ceylon leech are shown in Figure 2. D 2000Marker; 1 to 11: 1 to 14 represent the 14 primer combinations in Table 1;

[0026] Figure 3 The results of the condition optimization test of Arugam Ceylon leech in Example 2; A: ERA-LFD temperature optimization test results; B: ERA-LFD time optimization test results; NTC: negative control;

[0027] Figure 4 The results of sensitivity test of Hirudo aculeatus in Arugam in Example 3 are as follows: A: ERA-LFD sensitivity test result; B: conventional PCR sensitivity test result; NTC: negative control;

[0028] Figure 5 The specific detection results of Hirudo aculeatus in Arugam in Example 4; A: conventional PCR specificity results; B: ERA-LFD specificity test results; NTC: negative control;

[0029] Figure 6 The results of conventional PCR testing of 44 samples of grouper and golden pomfret collected in Hainan using the Arugam Ceylon leech were obtained using the method of Example 5;

[0030] A: 1 to 14 correspond to the 14 samples of pearl grouper in "N1 to N14" in Table 2; 15 to 20 correspond to the 6 samples of golden pomfret in "N15 to N20" in Table 2;

[0031] B: 21-36, 44 correspond to the 17 golden pomfret samples in Table 2 "N21-N36, N44" respectively; 37-42 correspond to the 6 leopard gill perch samples in Table 2 "N37-N42" respectively; 43 corresponds to the 1 blue spot sample in Table 2 "N43";

[0032] C: 45 to 63 correspond to the 19 water samples in Table 3 “W45 to W63” respectively. DETAILED DESCRIPTION

[0033] To achieve the purpose of the present invention, the Latin name of the Arugam Ceylon leech is Zeylanicobdella arugamensis; the method comprises detecting the COI gene sequence of the Arugam Ceylon leech using the enzymatic isothermal amplification technique ERA;

[0034] The primer sequences of the ERA are: ZE-PAGE-F1; ZE-HPLC-R1; in the direction from the 5' end to the 3' end, the nucleotide sequence of the ZE-PAGE-F1 is sequence number (ID): 1; the nucleotide sequence of the ZE-HPLC-R1 is sequence number (ID): 2.

[0035] In a specific embodiment, the ZE-PAGE-F1 primer is purified by polyacrylamide gel electrophoresis, and the ZE-HPLC-R1 primer is purified by high performance liquid chromatography.

[0036] In a specific embodiment, the length of the primer is 220 bp.

[0037] In one embodiment, the ERA is an ERA combined with a lateral flow chromatography test strip.

[0038] In a specific embodiment, the ERA probe is a ZE-HPLC-probe; in the direction from the 5' end to the 3' end, the nucleotide sequence of the ZE-HPLC-probe is sequence number (ID): 15; a FAM fluorescent group is set at the 5' end of sequence number (ID): 15, and a C3-Spacer blocking group is set at the 3' end, and a base located 30 bp downstream of the FAM fluorescent group and 15 bp upstream of the C3-Spacer blocking group is replaced with tetrahydrofuran.

[0039] In a specific embodiment, the probe is 47 bp in length.

[0040] In a specific embodiment, the primer for the ERA-binding lateral flow chromatography test strip is ZE-HPLC-R1-biotin, and the ZE-HPLC-R1-biotin is labeled with vitamin B7 at the 5' end of ZE-HPLC-R1.

[0041] In a specific embodiment, the reaction temperature of the ERA combined with the lateral flow chromatography test strip is 39° C. to 45° C.; and the reaction time is 5 to 30 minutes.

[0042] In a specific embodiment, the reaction temperature of the ERA combined with the lateral flow chromatography test strip is 41° C. and the reaction time is 10 min.

[0043] In one embodiment, the detection limit of the ERA is 1.6×10 1 copies / μL.

[0044] The specific embodiments of the present invention are further described below in conjunction with examples, but the present invention is not limited to the scope of the examples.

[0045] 1.1 Main Materials

[0046] Pathogenic materials include Arugam Ceylon leech, Vibrio harveyi, Vibrio alginolyticus, Vibrio parahaemolyticus, Singapore grouper iridovirus, Photobacterium damselae subsp. damselae (PDD), Enterospora epinepheli, Amyloodinium ocellatum, Cryptocaryon irritans Brown, grouper Nervous Necrosis Virus (NNV) cDNA, and Streptococcus iniae, all of which were tested and preserved in our laboratory.

[0047] The clinical samples and experimental materials for this study were collected from various farms in Hainan Province, including leopard gill bass (Plectropomus leopardus), pearl grouper (Epinephelus fuscoguttatus♀×E. lanceolatus♂), golden pomfret (Trachinotus ovatus), and blue grouper (Epinephelus awoara). See Tables 2 and 3 for details. The collection of these samples provided important evidence for the detection and analysis of Arugam's Ceylon leech. Photos of some typical symptoms of infected fish are shown below. Figure 1 shown.

[0048] 1.2 Main Reagents

[0049] Basic freeze-dried microspheres (ERA method), probe freeze-dried microspheres (ERA method), directional flow test strips (Suzhou Xianda Company, China), FastPure Gel DNA Extraction Mini Kit DC301-01 gel recovery / DNA purification kit (Nanjing Novozymes Biotech Co., Ltd., China), tissue genomic DNA extraction kit (Omega Bio-Tek, USA), water genomic DNA extraction kit (Omega Bio-Tek, USA).

[0050] Example 1

[0051] There are 14 primer combinations. Polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography (HPLC) were selected for primer purification. The names "PAGE" and "HPLC" represent different primer purification methods, respectively. The primer combination for conventional PCR is ZE-F / ZE-R.

[0052] The dipstick probe has a FAM fluorescent group at the 5' end and THF added at the 30th base from the 5' end. It also has a C3-Spacer blocking group at the 3' end, with one base replaced. Primer and probe information is shown in Table 1.

[0053] Table 1 Primer and probe information

[0054]

[0055]

[0056] Template DNA extraction

[0057] Total DNA was extracted from the tissues according to the instructions of the EZNATissue DNA Kit D3396 kit from OMEGA. Approximately 30 mg of DNA was extracted from each sample. 60 μL of preheated Elution Buffer was added to the center of the adsorption column, and the eluted DNA was stored at -20°C.

[0058] For DNA extraction from water samples, the water sample (3 L) was first filtered using microporous filter paper (pore size 0.22 μm). The filtered filter paper was cut into pieces and placed in a clean 50 mL centrifuge tube. The total water sample DNA was then extracted according to the instructions of the EZNA Water DNA Kit D5525-01 kit from OMEGA. The DNA concentration was determined using a Nanodrop microspectrophotometer, and the eluted DNA was stored at -20°C.

[0059] Construction and synthesis of positive plasmid standards

[0060] Using DNA from Hirugam ceylonii as a template, the COI gene fragment of Hirugam ceylonii was amplified using primer combination No. 17 in Table 1. The electrophoresis band was observed to determine whether it was at the expected product length of 611 bp. After confirmation, the amplified fragment containing the target gene was gel purified using the FastPureGel DNA Extraction Mini Kit DC301-01 (Nanjing Novozymes Biotech Co., Ltd., China).

[0061] The purified product was ligated into the PEASY-T1Sample vector CT111 (Beijing Quanshijin Biotechnology Co., Ltd., China) to construct a recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli DH5α competent cells ZC101 (Shanghai Weidi Biotechnology Co., Ltd., China) and plated onto LB plates containing ampicillin and incubated at 37°C for 12–16 h. After single colonies grew, positive colonies were selected and diluted in enzyme-free sterile water. M13 vector primers and colony PCR were used for screening. A portion of the expected positive clones were sequenced and confirmed, while the remaining portion was inoculated into LB liquid medium and cultured overnight. Plasmids were then extracted using the Omega Plasmid Mini Kit I Plasmid DNA Extraction Kit D6943 (Omega Bio-Tek, USA). The plasmid DNA concentration was determined to be 222 ng / μL using a Shanghai Instruments spectrophotometer. The plasmid concentration was calculated based on the Avogadro constant to be 4.56 × 10 10 copies / μL, diluted to 1.6×10 10 The copies / μL were stored at -20°C for subsequent ERA detection.

[0062] Basic ERA test

[0063] Basic ERA was performed using basic lyophilized microspheres (Suzhou Xinda Gene Technology Co., Ltd., China). The basic ERA reaction system consisted of 2.1 μL of 10 μM forward primer, 2.1 μL of 10 μM reverse primer, 2 μL of template DNA, and 41.8 μL of enzyme-free water, making up a 48 μL premix. First, 48 μL of the premix was added to each tube of amplification reagent. Then, 2 μL of ERA activator was added to the tube cap, and the mixture was quickly centrifuged and mixed. To screen for the optimal primer combination, a basic ERA reaction was performed at 45°C for 20 min using each primer combination listed in Table 1. After the reaction, 2.5 μL of Protain K was added for product purification and gel verification. Primers with high brightness and no primer-dimer bands were selected for subsequent experiments.

[0064] ERA-LFD detection of Arugam Ceylon leech

[0065] Before the experiment, the ERA-LFD kit enzyme freeze-dried powder should be removed and placed at room temperature for at least 10 minutes. ERA-LFD uses probe-type freeze-dried microspheres (ERA method) KS302 and flow test strips TS101 (Suzhou Xianda Gene Technology Co., Ltd., China).

[0066] The primers and probes used for ERA-LFD are shown in Table 1. To a tube containing lyophilized enzyme powder, add 2.1 μL of forward primer (10 μM), 2.1 μL of reverse primer (10 μM), 0.6 μL of probe (10 μM), and 41.2 μL of enzyme-free water to create a 46 μL premix. Then, add 2 μL of ERA activator to the tube cap. Add 2 μL of DNA template to the amplification zone, quickly centrifuge, and mix thoroughly. After incubation in a 45°C water bath for 10 minutes, the reaction is monitored using a test strip. The amplified product is diluted with enzyme-free water at a 60:1 dilution ratio (i.e., transfer 5 μL of the reaction product to a 1.5 mL centrifuge tube and dilute with 295 μL of pure water). Aspirate 80 μL into a new 1.5 mL centrifuge tube, insert the test strip into the tube, and read the results after 7–10 minutes. Ensure the control line is blue, and the intensity of the reaction is determined by the depth of the red color of the test line. In the ERA primer screening experiment, using 10 ng / μL Arugam Ceylon leech DNA as a template, all 14 primer combinations listed in Table 1 successfully amplified the target sequence. On the agarose gel, the positive amplification bands were observed, and the primers with higher brightness or no primer dimer bands were selected for subsequent experiments. The experimental results are shown in Figure 1. Figure 2 Primer combination number 1 (ZE-PAGE-1 / ZE-PAGE-R1) is the best, and the target product amplified by it is 220 bp in length.

[0067] In the ERA-LFD experiment, the reverse primer was purified better by "HPLC". In the following ERA-LFD experiment, the primer and probe combination used in the ERA-LFD method was the combination 15 listed in Table 1.

[0068] Example 2

[0069] ERA-LFD has a 1.6×10 5 The recombinant plasmid standard with 100 copies / μL was used as the positive template. The reaction was carried out at 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, and 45°C for 10 minutes. After the reaction, 5 μL of the reaction solution was mixed in 295 μL of enzyme-free water. 80 μL was then aspirated into a new 1.5 mL centrifuge tube and tested with a test strip. The experimental results are shown in the figure below. Figure 3As shown in A, the red color of the control bands at 41℃, 43℃ and 45℃ is very obvious, so the lowest temperature of 41℃ is selected as the optimal reaction temperature for the subsequent reaction. With 41℃ as the reaction temperature, the reaction was carried out for 5min, 10min, 15min, 20min, 25min and 30min respectively. The results are shown in Figure 3 B. The red color of the control was very weak at 5 min. During 10-20 min, the red color of the control was very obvious and the color intensity did not increase any more. The color weakened at 25 and 30 min. The optimal reaction time was 10 min.

[0070] Example 3

[0071] Sensitivity test

[0072] Gradual dilution of recombinant plasmid standard, with a concentration gradient of 1.6×10 0 ~1.6×10 8 Recombinant plasmids at 50 copies / μL were used as templates for ERA-LFD reactions, and the results were compared with those of conventional PCR. The ERA-LFD reaction system consisted of 2.1 μL of 10 μM forward primer, 2.1 μL of 10 μM reverse primer, 2 μL of template DNA, 0.6 μL of probe, 41.2 μL of enzyme-free water, and 2 μL of activator. Reaction conditions were 41°C for 10 min. Conventional PCR reaction system consisted of 12.5 μL of Taq Master Mix, 9.5 μL of ddH2O, 1 μL of forward primer, 1 μL of reverse primer, and 1 μL of DNA template. The reaction procedure was 30 cycles of 95°C for 3 min, 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, and 72°C for 5 min.

[0073] ERA-LFD test results are as follows Figure 4 As shown in A, at a concentration of 1.6×10 1 ~1.6×10 8 The test strips in the test line of the copies / μL range all showed red bands, so the minimum detection limit of the ERA-LFD method established in this study was 1.6×10 1 copies / μL. Conventional PCR test results are as follows Figure 4 As shown in B, at a concentration of 1.6×10 8 ~1.6×10 3 The bands were displayed in the copies / μL range, and the lowest detection limit was 1.6×10 3 copies / μL.

[0074] Example 4

[0075] The established ERA-LFD and conventional PCR methods were used to specifically detect important marine fish pathogens using DNA from Arugam leech, Vibrio harveyi, Vibrio alginolyticus, Vibrio parahaemolyticus, grouper iridovirus, Photorhabdus mermanii, Enterospora grouper, dinoflagellate amylovora, Cryptocaryon irritans, grouper neuronecrosis virus cDNA, and Streptococcus iniae as templates. The conventional PCR reaction protocol was: 95°C for 3 minutes, 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, and 72°C for 5 minutes, repeated 30 times. The reaction system consisted of 12.5 μL Taq Master Mix, 9.5 μL ddH₂O, 1 μL forward primer, 1 μL reverse primer, and 1 μL DNA template. The reaction system for ERA-LFD sensitivity detection is as follows: 2.1 μL of forward primer (10 μM), 2.1 μL of reverse primer (10 μM), 2 μL of template DNA, 0.6 μL of probe, 41.2 μL of enzyme-free water, and 2 μL of activator. Reaction conditions: 43°C, 10 min.

[0076] The results are as follows Figure 5 A and Figure 5 As shown in B, only the test strip test line and gel running results using the Arugam Ceylon leech sample as a template showed red bands and amplified bands, respectively. Other pathogens and negative controls all showed negative results, indicating that the two established methods have good specificity for Arugam Ceylon leech.

[0077] From the above, it can be seen that the present invention does not need to rely on complex instruments such as PCR instruments, and can complete the detection through constant temperature amplification, which has the advantages of simple operation, short reaction time and high sensitivity. The ERA primer combination ZE-PAGE-F1 / ZE-HPLC-R1 primer of the present invention is better than the other 13 primer groups. Under the constant temperature of 41°C, the results can be obtained within 10 minutes by the ERA-LFD detection method, and the detection sensitivity is as high as 1.6×101copies / μL, which is significantly better than the conventional PCR method. At the same time, specificity experiments show that the ERA-LFD detection method has good specificity for Arugam Ceylon leech and has no cross-reaction with other common pathogens of marine fish.

[0078] The rapid detection method of ERA-LFD of the present invention provides an efficient tool for early diagnosis and effective prevention and control of Arugam Ceylon leech.

[0079] Example 5

[0080] A total of 44 tissue samples and 19 water samples collected in 2024 were tested for Arugam Ceylon leech using the ERA-LFD detection method of Example 3 together with the basic ERA and conventional PCR detection methods to evaluate the application potential of ERA-LFD in clinical samples.

[0081] The positive results of conventional PCR, basic ERA, and ERA-LFD were counted (Table 4). Specific gel images of each detection method: Among them, the conventional PCR method detected 21 positive samples: 6 tissue samples from Lingao City, 8 tissue samples from Haikou City, and 7 tissue samples from Danzhou City; 2 positive water samples from Lingao City, 1 from Danzhou City, and 1 from Dongfang City. The results are shown in Figure 6 A. The basic ERA and ERA-LFD methods detected 26 positive samples: 9 tissue samples from Lingao City, 10 tissue samples from Haikou City, and 7 tissue samples from Danzhou City; 4 positive water samples from Lingao City, 2 from Danzhou City, and 2 from Dongfang City ( Figure 6 B and Figure 6 C).

[0082] Table 2. Detection results of conventional PCR, basic ERA, and ERA-LFD on clinical fish tissue samples

[0083]

[0084]

[0085]

[0086] Note: - represents the negative result of the Arugam Ceylon leech test; + represents the positive result of the Arugam Ceylon leech test

[0087] Table 3. Detection results of environmental water samples by conventional PCR, basic ERA, and ERA-LFD

[0088]

[0089] Note: - represents the negative result of the Arugam Ceylon leech test; + represents the positive result of the Arugam Ceylon leech test

[0090] Table 4 Statistics of the detection results of Arugam Ceylon leech by basic ERA, ERA-LFD and conventional PCR

[0091]

[0092] Positive rates for tissue and water samples showed that ERA-LFD and basic ERA were consistent, with a positive rate of 59.1% for Arugam Ceylon leech in tissue samples and 42.1% in water samples. Conventional PCR showed positive rates of 47.7% for tissue samples and 21.1% for water samples. This further validated the high sensitivity and reliability of the ERA-LFD method and demonstrated its ability to meet the needs of rapid diagnosis in practical production.

Claims

1. A method for detecting the Arugam Ceylon leech, characterized in that: The Latin name of the Arugam Ceylon leech is Zeylanicobdella arugamensis. The method includes detecting the COI gene sequence of the Arugam Ceylon leech using an enzymatic isothermal amplification (ERA) technique. The primer sequences of the ERA are: ZE-PAGE-F1; ZE-HPLC-R1. From the 5' end to the 3' end, the nucleotide sequence of the ZE-PAGE-F1 is sequence number (ID): 1; the nucleotide sequence of the ZE-HPLC-R1 is sequence number (ID):

2.

2. The method for detecting the leech Arugam Ceylon according to claim 1, wherein: The purification method of the ZE-PAGE-F1 primer is polyacrylamide gel electrophoresis, and the purification method of the ZE-HPLC-R1 primer is high performance liquid chromatography purification.

3. The method for detecting the leech Arugam Ceylon according to claim 1 or 2, wherein: The length of the primer is 220 bp.

4. The method for detecting the leech Arugam Ceylon according to claim 1 or 2, wherein: The ERA is an ERA combined with a lateral flow chromatography test strip.

5. The method for detecting the leech of Arugam Ceylon according to claim 4, wherein: The ERA probe is a ZE-HPLC-probe; from the 5' end to the 3' end, the nucleotide sequence of the ZE-HPLC-probe is sequence number (ID): 15; a FAM fluorescent group is set at the 5' end of sequence number (ID): 15, and a C3-Spacer blocking group is set at the 3' end, and a base located 30 bp downstream of the FAM fluorescent group and 15 bp upstream of the C3-Spacer blocking group is replaced with tetrahydrofuran.

6. The method for detecting the leech Arugam Ceylon according to claim 5, characterized in that: The probe is 47 bp in length.

7. The method for detecting the leech Arugam Ceylon according to claim 4, wherein: The primer of the ERA combined with lateral flow chromatography test strip is ZE-HPLC-R1-biotin, and the ZE-HPLC-R1-biotin is labeled with vitamin B7 at the 5' end of ZE-HPLC-R1.

8. The method for detecting the Arugam Ceylon leech according to claim 4, wherein: The reaction temperature of the ERA combined with the lateral flow chromatography test strip is 39° C. to 45° C., and the reaction time is 5 to 30 minutes.

9. The method for detecting the Arugam Ceylon leech according to claim 8, characterized in that: The reaction temperature of the ERA combined with the lateral flow chromatography test strip is 41° C. and the reaction time is 10 min.

10. The method for detecting the Arugam Ceylon leech according to claim 1 or 2, characterized in that: The detection limit of the ERA was 1.6×10 1 copies / μL.

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