Primer probe group and kit for species identification of echinococcus

Abstract

The invention relates to the technical field of molecular biology, and discloses a primer probe set for species identification of echinococcus granulosus, and the primer probe set comprises a first primer probe set for targeting echinococcus granulosus, a second primer probe set for targeting echinococcus multilocularis and / or a third primer probe set for targeting echinococcus stone. According to the primer probe group, synchronous, rapid and high-specificity identification and detection of three kinds of echinococcus, namely echinococcus granulosus (Eg), echinococcus multilocularis (Em) and echinococcus stone (Es), are realized. When the primer probe group is used for identifying and detecting the three kinds of echinococcus, the detection sensitivity reaches 200 copies / mL, and the positive coincidence rate reaches 100%. The primer probe group realizes synchronous and accurate identification of echinococcus species, and has a wide application prospect in molecular epidemiological investigation and port quarantine.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a primer and probe set and kit for species identification of Echinococcus tapeworm. Background Technology

[0002] Echinococcosis is an infectious parasitic disease caused by adult Echinococcus tapeworms parasitizing canines and other carnivorous animals. Echinococcosis is an important zoonotic disease caused by the larvae of Echinococcus tapeworms infecting humans or herbivores. The transmission cycle of Echinococcus tapeworms mainly takes three forms: the domestic animal cycle, the wild animal cycle, and a mixed type (wild-domestic). Carnivorous canines are an important source of infection for echinococcosis. Adult Echinococcus tapeworms parasitize the small intestine of carnivorous canines (definitive hosts). After the worm matures, its eggs are excreted in feces, contaminating food, water, and the surrounding environment, causing infection in humans or herbivores (intermediate hosts). This results in lesions in organs such as the liver and lungs of humans or livestock, making them patients or infected animals with echinococcosis. The predator-prey relationship between canines and herbivores maintains the transmission cycle of echinococcosis in nature. Data from the World Health Organization (WHO) shows that echinococcosis and echinococcosis are highly endemic in the Mediterranean coast, Eastern Europe, Central Asia, western South America, and pastoral areas of western my country. In particular, the prevalence rate in pastoral populations in western provinces such as Xinjiang, Qinghai, Tibet, and Sichuan in my country is as high as 3 to 10%.

[0003] Currently, the recognized Echinococcus tapeworms include *Echinococcus granulosus*, *Echinococcus multilocularis*, *Echinococcus shiquensis*, *Echinococcus fowleri*, and *Echinococcus septemlobus*. In my country, the three main prevalent species of Echinococcus tapeworms are *Echinococcus granulosus*, *Echinococcus multilocularis*, and *Echinococcus shiquensis*. Among them, *Echinococcus granulosus* (… Echinococcus granulosus Echinococcosis, caused by E. coli (E.g.), is the most widespread, occurring in over 100 countries worldwide; Echinococcus multilocularis (…) Echinococcus multilocularis Echinococcal cysticercosis caused by *Echinococcus scutellatus* is mainly distributed in the cold and temperate regions of the Northern Hemisphere, with a mortality rate exceeding 90%; *Echinococcus scutellatus* (…) Echinococcus shiquicus ,Es) is a species endemic to the Qinghai-Tibet Plateau, which can cause echinococcosis in Shiqu County. Its biological evolution, genetic and public health significance is becoming increasingly prominent.

[0004] Besides their distribution, the three species also exhibit significant differences in their host preferences and pathogenic mechanisms at different developmental stages. For example, during the larval stage, cystic echinococcosis primarily infects livestock (cattle, sheep, and humans), forming cystic lesions in organ tissues; alveolar echinococcosis prefers rodents and humans, presenting as infiltrative lesions; and *Echinococcus shiquensis* primarily infects plateau pikas, presenting as cystic lesions in the lungs. Since the adult stages of all three species can parasitize the small intestine of dogs and various other canids, traditional morphological examination methods involving necropsy to collect adult worms are necessary to determine infection status and the specific type of infection. This approach cannot achieve rapid and accurate species identification in domestic or wild canids. Therefore, it is necessary to establish rapid and accurate species identification methods for these three species to precisely monitor and control the source of infection, thereby limiting its spread in the human population.

[0005] Currently, the diagnosis of echinococcosis, an intermediate host disease, mainly relies on three methods: imaging examination, serological testing, and traditional PCR technology. Imaging examination is only suitable for human examinations, but it is difficult to implement in epidemiological screening of other intermediate hosts, and the complexity of differential diagnosis of disease types is high, easily leading to misdiagnosis. Currently available serological testing products cannot distinguish between species; their diagnostic sensitivity and specificity are poor due to the specificity of antigen components, and they suffer from cross-reactivity with other tapeworms. Furthermore, these tests are not easily applied in epidemiological screening of other intermediate hosts. While conventional PCR methods can improve specificity, they are cumbersome to operate and typically require 3-4 hours to obtain results, resulting in long processing times. In addition, most existing commercial kits for identifying Echinococcus tapeworm species based on conventional PCR methods are based on a single detection target, making it difficult to meet the clinical need for simultaneous identification and multiplex detection of multiple Echinococcus tapeworms.

[0006] Real-time quantitative PCR (qPCR) technology has become an important tool for molecular diagnostics due to its high sensitivity and specificity. Among them, TaqMan probe technology achieves specific detection through fluorescently labeled oligonucleotide probes, exhibiting higher specificity compared to the traditional SYBR Green method. Currently, some studies have attempted to use TaqMan and other qPCR technologies for species identification of Echinococcus tapeworms. However, this technology still has several limitations. For example, most studies still design primers and probes only for single species such as Eg or Em, which is insufficient to meet the needs of disease surveillance and clinical diagnosis for simultaneous identification of multiple Echinococcus tapeworms; signal interference exists between different fluorescence channels during multiplex detection; and there are few studies on the specific detection of Es, and the identification method is not yet fully standardized, which may affect the accuracy of the results. Therefore, there is an urgent clinical need to develop a high-efficiency detection kit based on TaqMan and other qPCR technologies that can simultaneously identify multiple Echinococcus tapeworms.

[0007] At the technical level, the main challenges facing multiplex real-time quantitative PCR include primer dimer formation, balancing different amplification efficiencies, and cross-interference of fluorescence channels. Especially when detecting complex samples (such as feces, tissues, etc.), the presence of inhibitors often leads to a decrease in detection sensitivity. These technical bottlenecks limit the application of existing methods in primary healthcare institutions and on-site testing. Given the limitations of existing detection technologies, there is an urgent need in this field to develop a molecular diagnostic scheme with the following key performance indicators: (1) strong species-specific recognition ability, which can accurately identify three pathogens: Eg, Em, and Es; (2) high sensitivity, which can be used for the detection of low biomass samples; (3) good multiplex detection capability, which can realize the simultaneous analysis of single reaction systems; (4) simple operation, which is suitable for clinical promotion; (5) including a quality control system to ensure the reliability of results. Summary of the Invention

[0008] This invention provides a primer and probe set and kit for identifying Echinococcus tapeworm species, in order to solve the problem of not being able to simultaneously and efficiently identify multiple Echinococcus tapeworm species.

[0009] In a first aspect, the present invention provides a primer and probe set for species identification of Echinococcus tapeworms, the primer and probe set comprising a first primer and probe set targeting Echinococcus granulosus, a second primer and probe set targeting Echinococcus multilocularis, and / or a third primer and probe set targeting Echinococcus shiquensis.

[0010] In one optional embodiment, the first primer-probe set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.1, a reverse primer with a nucleotide sequence as shown in SEQ ID NO.2, and a probe with a nucleotide sequence as shown in SEQ ID NO.3.

[0011] In one optional embodiment, the second primer-probe set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.4, a reverse primer with a nucleotide sequence as shown in SEQ ID NO.5, and a probe with a nucleotide sequence as shown in SEQ ID NO.6.

[0012] In one optional embodiment, the third primer-probe set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.7, a reverse primer with a nucleotide sequence as shown in SEQ ID NO.8, and a probe with a nucleotide sequence as shown in SEQ ID NO.9.

[0013] In one alternative implementation, the probe comprises a TaqMan fluorescent probe.

[0014] In one alternative embodiment, the probe is labeled with a fluorescent group and a quenching group.

[0015] In one alternative embodiment, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0016] In one alternative embodiment, the fluorescent group includes at least one of FAM, VIC, and ROX.

[0017] In one alternative embodiment, the quenching group includes MGB.

[0018] Secondly, the present invention also provides a kit for species identification of Echinococcus tapeworm, the kit comprising the above-mentioned primer and probe set.

[0019] In one alternative embodiment, the kit further includes DNA polymerase, dNTPs, and reaction buffer.

[0020] Thirdly, the present invention also provides a method for identifying the species of Echinococcus tapeworm, wherein the method is not for the purpose of disease diagnosis and treatment, and the method includes: using the above-mentioned kit to test the sample.

[0021] In one alternative implementation, the method includes the following steps: Step 1: Extract DNA from the sample to be tested; Step 2: Perform multiplex fluorescent PCR amplification on the extracted DNA using the above kit; Step 3: Identify the species of Echinococcus tapeworm in the sample by interpreting the Ct value.

[0022] In one alternative implementation, the sample to be tested is selected from feces, tissue, or environmental samples.

[0023] Fourthly, the present invention also provides the application of the above-mentioned primer and probe set, the above-mentioned kit, or the above-mentioned species identification method in identifying the species of Echinococcus tapeworm or in preparing products for diagnosing parasitic diseases.

[0024] In one optional embodiment, the parasitic disease includes echinococcosis caused by adult Echinococcus granulosus, cystic echinococcosis caused by larvae of Echinococcus granulosus, multilocular echinococcosis caused by adult Echinococcus multilocularis, alveolar echinococcosis caused by larvae of ...

[0025] The technical solution of this invention has the following advantages: 1. The present invention provides a primer and probe set for species identification of Echinococcus tapeworms, the primer and probe set comprising a first primer and probe set targeting Echinococcus granulosus, a second primer and probe set targeting Echinococcus multilocularis, and / or a third primer and probe set targeting Echinococcus shiquensis; the first primer and probe set comprising a forward primer with a nucleotide sequence as shown in SEQ ID NO.1, a reverse primer with a nucleotide sequence as shown in SEQ ID NO.2, and a probe with a nucleotide sequence as shown in SEQ ID NO.3; the second primer and probe set comprising a forward primer with a nucleotide sequence as shown in SEQ ID NO.4, a reverse primer with a nucleotide sequence as shown in SEQ ID NO.5, and a probe with a nucleotide sequence as shown in SEQ ID NO.6; the third primer and probe set comprising a forward primer with a nucleotide sequence as shown in SEQ ID NO.7, a reverse primer with a nucleotide sequence as shown in SEQ ID NO.8, and a probe with a nucleotide sequence as shown in SEQ ID NO.9. The primer and probe sets have the following advantages: (1) All three primer and probe sets have been rigorously verified by experiments and have no cross-reaction with common parasites, ensuring interspecificity; (2) The three primer and probe sets together achieve simultaneous, rapid, and highly specific identification and detection of three types of Echinococcus tapeworms: Echinococcus granulosus (Eg), Echinococcus multilocularis (Em), and Echinococcus shiquensis (Es); (3) The detection sensitivity of the three primer and probe sets for Eg, Em, and Es reaches 200 copies / mL, and the positive concordance rate reaches 100%.

[0026] 2. The present invention also provides a kit for species identification of Echinococcus tapeworms, the kit comprising the above-mentioned primer and probe set. The primer and probe set used in the kit has the following advantages: (1) all three primer and probe sets have been rigorously experimentally verified and have no cross-reactivity with common parasites, ensuring interspecific specificity; (2) the three primer and probe sets together achieve simultaneous, rapid, and highly specific identification and detection of three types of Echinococcus tapeworms: Echinococcus granulosus (Eg), Echinococcus multilocularis (Em), and Echinococcus steganatum (Es); (3) the detection sensitivity of the three primer and probe sets for Eg, Em, and Es of the three types of Echinococcus tapeworms all reaches 200 copies / mL, and the positive concordance rate reaches 100%.

[0027] 3. This invention also provides a method for species identification of Echinococcus tapeworms, using the aforementioned kit to detect the sample. The method boasts advantages such as high specificity, high sensitivity (detection limit up to 200 copies / mL), and good repeatability. It is also simple to operate, requiring minimal equipment and personnel expertise, and can complete the detection within one hour. It is suitable for clinical diagnosis, epidemiological investigations, and rapid on-site screening. Furthermore, the method exhibits no cross-reactivity with eight common parasites, and its amplification efficiency remains consistently within the optimized range of 92.58%–107.31%, meeting the technical specifications for molecular diagnostic reagents. This technological breakthrough provides a novel solution for the clinical diagnosis and treatment of echinococcosis and for epidemic prevention and control. Attached Figure Description

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 This is a flowchart of the detection method of the present invention; Figure 2 This is a specific test result for Echinococcus granulosus (Eg); Figure 3 This is a specific test result for Echinococcus multilocularis (Em); Figure 4 This is a specific test result for Echinococcus shiquensis (Es); Figure 5 These are the qPCR gradient dilution amplification curves and amplification efficiency results for Echinococcus granulosus (Eg). Figure 6 These are the qPCR gradient dilution amplification curves and amplification efficiency results for Echinococcus multilocularis (Em). Figure 7 These are the qPCR gradient dilution amplification curves and amplification efficiency results for Echinococcus shiquense (Es). Detailed Implementation

[0030] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0031] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0032] The primer and probe sets involved in the following examples were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; enzyme-free water, 10×PCR buffer, dNTPs, MgCl2, and Taq enzyme were purchased from Sangon Biotech (Shanghai) Co., Ltd.; 10×PCR buffer: 100 mM Tris-HCl (pH 8.9), 500 mM KCl. The positive references L1-L3 (positive controls for detection) involved in the following examples are inactivated engineered Escherichia coli containing the target fragment (the target fragment contains the gene sequence of the corresponding detection target). The target fragment of positive reference L1 (positive control for Echinococcus granulosus detection) contains the nucleotide sequence shown in SEQ ID NO.10; the target fragment of positive reference L2 (positive control for Echinococcus multilocularis detection) contains the nucleotide sequence shown in SEQ ID NO.11; and the target fragment of positive reference L3 (positive control for Echinococcus shiquensis detection) contains the nucleotide sequence shown in SEQ ID NO.12. Positive references L1-L3 were synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd. Nucleic acid samples infected with pork tapeworm, beef tapeworm, Spirometra ovalis, Clonorchis sinensis, Schistosoma japonicum, Giardia lamblia, Toxoplasma gondii, and Cryptosporidium microsporum were provided by Suzhou Zhongke Sujing Biotechnology Co., Ltd.

[0033] Example 1: A primer and probe set for species identification of Echinococcus tapeworm This embodiment provides a primer and probe set for species identification of Echinococcus tapeworms. The primer and probe set consists of a first primer and probe set targeting Echinococcus granulosus, a second primer and probe set targeting Echinococcus multilocularis, and a third primer and probe set targeting Echinococcus shiquensis. The first primer and probe set consists of a forward primer Eg-F with the nucleotide sequence shown in SEQ ID NO.1, a reverse primer Eg-R with the nucleotide sequence shown in SEQ ID NO.2, and a probe Eg-P with the nucleotide sequence shown in SEQ ID NO.3. The second primer and probe set consists of a forward primer Em-F with the nucleotide sequence shown in SEQ ID NO.4, a reverse primer Em-R with the nucleotide sequence shown in SEQ ID NO.5, and a probe Em-P with the nucleotide sequence shown in SEQ ID NO.6. The third primer and probe set consists of a forward primer Es-F with the nucleotide sequence shown in SEQ ID NO.7, a reverse primer Es-R with the nucleotide sequence shown in SEQ ID NO.8, and a probe Eg-P with the nucleotide sequence shown in SEQ ID NO.6. The probe Es-P composition shown in NO.9 (primer and probe sequence is shown in Table 1).

[0034] Table 1 Primer and probe sequence

[0035] The probes Eg-P, Em-P, and Es-P are labeled with fluorescent groups FAM, VIC, and ROX at their 5' ends, respectively, and with a quencher group MGB at their 3' ends. These fluorescent and quencher groups form a fluorescence resonance energy transfer (FRET) pair, achieving fluorescence quenching when the probes are intact. When the probes are cleaved by the 5'→3' exonuclease activity of Taq DNA polymerase, the fluorescent and quencher groups separate, generating a detectable fluorescent signal. The fluorescent probe labeling system is shown in Table 2.

[0036] Table 2 Fluorescent probe labeling system

[0037] Example 2: A kit for species identification of Echinococcus tapeworm This embodiment provides a kit for species identification of Echinococcus tapeworm. The kit consists of the primer and probe set described in Example 1, 10×PCR buffer, dNTPs, MgCl2, Taq enzyme, and enzyme-free water. The kit also includes a negative control and a positive control. The concentration of the dNTPs is 10 mmol / L, the concentration of the MgCl2 is 25 mmol / L, the concentration of the Taq enzyme is 5 U / μL, and the concentration of the primer-probe set is 10 μmol / L. The 10×PCR buffer consists of 100 mM Tris-HCl (pH 8.9) and 500 mM KCl.

[0038] The negative control was enzyme-free water, and the positive control was inactivated Escherichia coli engineered bacteria containing the target fragment. Specifically, the target fragment of positive reference L1 (positive control for Echinococcus granulosus detection) contained the nucleotide sequence shown in SEQ ID NO. 10; the target fragment of positive reference L2 (positive control for Echinococcus multilocularis detection) contained the nucleotide sequence shown in SEQ ID NO. 11; and the target fragment of positive reference L3 (positive control for Echinococcus shiquensis detection) contained the nucleotide sequence shown in SEQ ID NO. 12.

[0039] Example 3: A method for species identification of Echinococcus tapeworm This embodiment provides a method for species identification of Echinococcus tapeworms. The method uses the kit from Example 2 and specifically includes the following steps (see the flowchart of the detection method). Figure 1 ): (1) Tissue sample pretreatment: According to the instructions of the blood / cell / tissue genomic DNA extraction kit (Tiangen Biotech, DP304), liver tissue samples or fecal samples were prepared into homogenates for testing. (2) Extracting DNA from the sample to be tested: According to the instructions, take 200 μL of the sample homogenate obtained in step S01, digest the tissue cells with proteinase K (56℃) until the solution is clear, purify the DNA through a silica membrane adsorption column, and elute with 50 μL of LTE buffer to obtain the DNA template. (3) Configure the reaction system according to Table 3; (4) Add the DNA template obtained in step S02 to the reaction system described in step (3), and place it in a real-time fluorescence quantitative PCR instrument for amplification. The PCR amplification program is shown in Table 4. (5) Determine whether the sample to be tested contains Echinococcus tapeworm and the corresponding species of Echinococcus tapeworm based on the PCR detection results. The identification criteria are shown in Table 5.

[0040] Table 3 Reaction System

[0041] Table 4 PCR Amplification Program

[0042] Table 5. Criteria for determining the presence of Echinococcus tapeworms and their species in the test samples.

[0043] Examples 4-5: A method for species identification of Echinococcus tapeworms This embodiment provides a method for species identification of Echinococcus tapeworm. The method is based on Example 3, but the final concentration of MgCl2 in the reaction system is adjusted from 3mM to 4mM or 5mM, that is, the reaction system is adjusted as shown in Table 6 or Table 7. Other experimental steps are the same as in Example 3.

[0044] Table 6 Reaction System

[0045] Table 7 Reaction System

[0046] Experimental Example 1: Verification of the detection efficiency of the reaction system This experimental example verifies the detection efficacy of the reaction system in the identification methods described in Examples 3-5. The specific steps are as follows: Experiment 1: Verification of the detection efficiency of the reaction systems in Examples 3 and 4 (1) The target fragment was synthesized into a recombinant plasmid containing the sequence shown in SEQ ID NO.10~12. The plasmid vector was pUC57, and the recombinant plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd. (2) The absorbance at a wavelength of 260 nm was measured by spectrophotometry, and the concentration of the synthesized plasmid was calculated. Each recombinant plasmid was diluted 10-fold with enzyme-free water to a concentration of 2000 copies / mL and 200 copies / mL. Then, 5 μL of the diluted recombinant plasmid was taken and qPCR was performed according to the methods described in Examples 3-4, with 3 replicates for each concentration.

[0047] The experimental results are shown in Table 8. The results show that the positive detection rates of the reaction systems in Examples 3 and 4 are all high. Among them, compared with the reaction system described in Example 4 (before optimization), the reaction system described in Example 3 (after optimization) has higher detection efficiency and a positive detection rate of 100%, which proves that it has better detection sensitivity and repeatability.

[0048] Table 8. Validation of detection efficiency before and after PCR reaction system optimization

[0049] Experiment 2: Verification of the detection efficiency of the reaction systems in Examples 3-5 (1) The target fragment was synthesized into a recombinant plasmid containing the sequence shown in SEQ ID NO.10~12. The plasmid vector was pUC57, and the recombinant plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd. (2) The absorbance at a wavelength of 260 nm was measured by spectrophotometry, and the concentration of the synthesized plasmid was calculated. Each recombinant plasmid was serially diluted 10-fold with enzyme-free water to dilute the plasmid concentration to 10. 7 10 6 10 5 10 4 and 10 3 copies / mL, then take 5 μL and dilute to 10. 7 10 6 10 5 10 4 and 10 3 Recombinant plasmids of copies / mL were used, with enzyme-free water as a negative control, and qPCR reactions were performed according to the methods described in Examples 3-5.

[0050] The experimental results are shown in Table 9. The results show that the reaction systems used in Examples 3-5 can be detected in reaction systems at various dilutions. Combined with the results obtained in Experiment 1, it can be seen that the magnesium ion concentration in the reaction system has a certain impact on the detection sensitivity. At the magnesium ion concentration used in Example 3, the detection sensitivity of the detection system is slightly higher.

[0051] Table 9. Amplification effect under different reaction systems and concentrations

[0052] Experimental Example 2: Specific Detection of Primer-Probe Sets This experimental example demonstrates the specificity of the primer-probe set described in Example 1 through a cross-reactivity assay. The specific steps are as follows: (1) Eight parasites were selected, including Taenia solium, Moniezia spp., Spirometra erinaceieuropaei, Clonorchis sinensis, Schistosoma japonicum, Giardia lamblia, Toxoplasmagondii, Cryptosporidium parvum, as well as Echinococcus granulosus (Eg), Echinococcus multilocularis (Em) and Echinococcus shiquensis (Es), as samples to be tested. (2) Dilute the sample DNA from step (1) to a concentration of 1×10⁻⁶. 5The samples were tested at 1000 copies / mL, and a template-free negative control (NTC) was set up. The reaction system was configured and the amplification program was set according to the experimental method in Example 3. Two replicates were set for each sample, and the samples were tested by real-time fluorescence quantitative PCR. (3) By monitoring the signal values ​​of the three fluorescence channels FAM (Eg), VIC (Em) and ROX (Es), and taking Ct value <37 and typical amplification curve as the positive standard, it is determined whether the sample contains Echinococcus tapeworm and the corresponding Echinococcus tapeworm species.

[0053] The results are as follows Figures 2-4 As shown, the results indicate that only the target pathogens (Eg, Em, Es) showed specific amplification in the corresponding detection channels, while 10 non-target parasites (8 other pathogens and 2 other tapeworms) and the negative control did not show effective amplification signals, proving that the primer and probe set described in this invention has high specificity for Echinococcus granulosus, Echinococcus multilocularis, and Echinococcus shiquensis.

[0054] Experiment Example 3: Verification of qPCR amplification efficiency of primer and probe sets This experimental example verifies the amplification efficiency of the primer and probe set described in Example 1 through a gradient dilution experiment. The specific steps are as follows: Genomic DNA was extracted from *Echinococcus granulosus* (Eg), *Echinococcus multilocularis* (Em), and *Echinococcus shiquensis* (Es), and 10-fold dilutions were used to prepare 10... 3 ~10 7 Template solutions with five concentration gradients (copies / mL) were prepared, and a template-free control (NTC) was also included. The reaction system and amplification program were configured according to the method described in Example 3.

[0055] The results are as follows Figures 5-7 As shown, the results indicate that all three target pathogens exhibit a favorable dose-response relationship: as the template concentration increases from 10... 3 copies / mL increased to 10 7 The copies / mL corresponded to Ct values ​​that gradually decreased from >30 to 15-16. Standard curve analysis showed amplification efficiencies of 92.58% (R²=0.9978), 107.31% (R²=0.9816), and 96.24% (R²=0.9997) for Eg, Em, and Es, respectively, all meeting the industry standard requirement of 90-110%. No specific amplification signal was detected in any NTC wells (Ct value >40), indicating that the primer-probe set described in Example 1 has excellent amplification performance.

[0056] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A primer and probe set for species identification of Echinococcus tapeworm, characterized in that, The primer and probe set includes a first primer and probe set targeting Echinococcus granulosus, a second primer and probe set targeting Echinococcus multilocularis, and / or a third primer and probe set targeting Echinococcus shiquensis.

2. The primer-probe set according to claim 1, characterized in that, The first primer-probe set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.1, a reverse primer with a nucleotide sequence as shown in SEQ ID NO.2, and a probe with a nucleotide sequence as shown in SEQ ID NO.

3.

3. The primer-probe set according to claim 1, characterized in that, The second primer-probe set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.4, a reverse primer with a nucleotide sequence as shown in SEQ ID NO.5, and a probe with a nucleotide sequence as shown in SEQ ID NO.

6.

4. The primer-probe set according to claim 1, characterized in that, The third primer-probe set includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.7, a reverse primer with a nucleotide sequence as shown in SEQ ID NO.8, and a probe with a nucleotide sequence as shown in SEQ ID NO.

9.

5. The primer-probe set according to any one of claims 1 to 4, characterized in that, The probe is labeled with fluorescent groups and quenching groups.

6. The primer-probe set according to claim 5, characterized in that, The probe is labeled with a fluorescent group at its 5' end and a quenching group at its 3' end; Optionally, the fluorescent group includes at least one of FAM, VIC, and ROX; Optionally, the quenching group includes MGB.

7. A kit for species identification of Echinococcus tapeworm, characterized in that, The kit comprises the primer and probe set as described in any one of claims 1 to 5.

8. The reagent kit according to claim 7, characterized in that, The kit also includes DNA polymerase, dNTPs, and buffer.

9. A method for species identification of Echinococcus tapeworm, wherein the method is not for the purpose of disease diagnosis and treatment, characterized in that, The method includes: testing the sample to be tested using the kit described in claim 7 or 8.

10. The use of the primer and probe set according to any one of claims 1 to 6, the kit according to any one of claims 7 to 8, or the species identification method according to claim 9 in identifying the species of Echinococcus tapeworm or in preparing products for diagnosing parasitic diseases.

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