Primer combination, kit and detection method for simultaneously detecting three echinococcosis pathogens

Abstract

The invention discloses a primer combination, a kit and a detection method for simultaneously detecting three echinococcosis pathogens, and belongs to the technical field of parasitic pathogen detection. The primer combination comprises a primer sequence as shown in SEQ ID NO. 1-6. The primer combination comprises a primer sequence as shown in SEQ ID NO. The primer pair combination designed by the invention does not interfere with each other when multiple PCR amplification is carried out in the same system under the same condition, is strong in specificity and high in sensitivity, and is designed for echinococcosis pathogens (echinococcus multilocularis, narrow-sense echinococcus granulosus and echinococcus stone). On the basis, a multiplex PCR method capable of identifying narrow-sense echinococcus granulosus, echinococcus multilocularis and echinococcus stone in faeces and tissues is established, the method is high in specificity and sensitivity, has the advantages of high flux, simplicity, convenience and rapidness, can remarkably improve the working efficiency, reduces the time and economic cost, and is suitable for popularization and application. The method has a wide application prospect in the field of echinococcosis molecular epidemiological investigation.

Description

Technical Field

[0001] This invention belongs to the field of parasitic pathogen detection technology, specifically relating to a primer combination, reagent kit, and detection method for simultaneously detecting three echinococcosis pathogens. Background Technology

[0002] Echinococcosis is a zoonotic parasitic disease caused by Echinococcus tapeworms. The main pathogens causing echinococcosis in my country are Echinococcus granulosus sensustricto, Echinococcus multilocularis, and Echinococcus shiquicus. The intermediate hosts of Echinococcus granulosus in the narrow sense include even-toed ungulates such as cattle and sheep, while its definitive host is the domestic dog. The intermediate hosts of Echinococcus shiquensis and Echinococcus multilocularis are various small mammals, such as rodents like the plateau pika (Ochotona curzoniae) and the Qinghai field mouse (Microtus fuscus), while their definitive hosts are wild canids such as wolves and foxes. Humans can contract echinococcosis (Echinococcus granulosus or Echinococcus multilocularis) by accidentally ingesting Echinococcus eggs, but humans are not infected with Echinococcus shiquensis.

[0003] Currently, the methods commonly used by disease control centers and hospitals to detect echinococcosis pathogens are immunological methods that detect antigens or antibodies. However, these methods are prone to false positives due to the presence of common antigenic determinants; furthermore, the results are often inaccurate and prone to false negatives due to variations in sample preservation conditions. While echinococcosis can also be diagnosed through imaging techniques such as ultrasound and CT scans, and pathological examinations, these methods present challenges in subtyping and are prone to misdiagnosis or missed diagnosis of small or atypical lesions.

[0004] Due to its speed, accuracy, and sensitivity, PCR technology is increasingly being used in the diagnosis and detection of various parasitic diseases. Currently, commonly used PCR techniques include singlet PCR, multiplex PCR, and nested PCR. Nakao M (Nakao M, Sako Y, Yokoyama N, et al. Mitochondrial genetic code in cestodes[J]. Mol BiochemParasitol, 2000, 111(2): 415-424.), Boufana B (Development of Three PCR Assays for the Differentiation between Echinococcus shiquicus, E. granulosus (G1genotype) and E. multilocularis DNA in the Co-Endemic Region of Qinghai-Tibet plateau, China[J]. American Journal of Tropical Medicine & Hygiene, 2013, 88(4): 795-802), Wang Huipin (Wang Huipin, Li Xin, Sun Shijun. Degenerate primer q-PCR method for detecting Echinococcus granulosus in sheep liver tissue in Kashgar, Xinjiang[J]. Guangdong Medical Journal, 2022, 43(01):46-50.DOI:10.13820 / j.cnki.gdyx.20211366.) and others have successively established single-pair PCR technology for detecting Echinococcus granulosus. However, single-pair PCR can only identify one species in a single reaction system. The detection procedure is cumbersome, time-consuming and labor-intensive, which is not conducive to the rapid detection of large-scale samples.

[0005] Multiplex PCR can simultaneously detect two or more different target genes in the same system and under the same reaction conditions, meaning that one reaction system can identify multiple species at the same time, which greatly improves the efficiency of the method. Chen Fan et al. (patent CN201710392941.8) established a multiplex PCR technique for simultaneously identifying Echinococcus granulosus G1-G3, Echinococcus multilocularis, and Echinococcus granulosus G6-G10 in lesion tissues; Shang Jingye et al. (patent CN201910022916.X) established a multiplex PCR technique that can be used to identify Echinococcus granulosus, Echinococcus cantonensis, and Echinococcus multilocularis in tissues; Lou Zhongzi et al. (patent CN201310102181.4) and Cong-Nuan Liu et al. (Liu CN, Lou ZZ, Li L, Yan HB, Blair D, Lei MT, Cai JZ, Fan YL, Li JQ, Fu BQ, Yang YR, McManus DP, Jia WZ. Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay. PLoS Negl Trop Dis. 2015 Sep 22;9(9):e0004084. doi: 10.1371 / journal.pntd.0004084. PMID: 26393793; PMCID:PMC4578771.) established a multiplex PCR method for the simultaneous detection of Echinococcus granulosus, Echinococcus shiquensis and Echinococcus multilocularis in canine feces.

[0006] However, in multiplex PCR detection, primer pairs are prone to cross-reaction with closely related pathogens, leading to false positive results. Furthermore, complementary binding between primer pairs can easily cause interference. Therefore, there is an urgent need to establish an efficient and convenient multiplex PCR method for identifying *Echinococcus granulosus*, *Echinococcus multilocularis*, and *Echinococcus shiquensis* in feces and tissues. Summary of the Invention

[0007] To address the aforementioned shortcomings in the existing technology, this invention provides a primer combination, kit, and detection method for simultaneously detecting three echinococcosis pathogens. The primer pairs designed in this invention do not interfere with each other when multiplex PCR amplification is performed in the same system and under the same conditions, exhibiting high specificity and sensitivity. They are specifically designed for echinococcosis pathogens (Echinococcus multilocularis, Echinococcus granulosus, and Echinococcus shiquensis).

[0008] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: A primer combination for simultaneous detection of three echinococcosis pathogens, comprising the following primer sequences: Em-F1: 5'-GTCAGTCTTGTTGTTGTGTT-3'; Em-R1: 5'-TACTCTAGTAGCCTCAGCAT-3'; Eg-F2: 5'-AGGTCGGTTCGATGTGCTTT-3'; Eg-R2: 5'-CCCGCTGACCAACTCTCTTT-3'; Es-F3: 5'-GAGAATGTTGTGATTAGGTAGG-3'; Es-R3: 5'-CGAAGAAGATAACGGAGGAT-3'.

[0009] A kit for simultaneously detecting three echinococcosis pathogens, comprising the primer combination described above.

[0010] Furthermore, the kit also includes Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, 2×Taq PCR amplification reagent with gel loading dye, and nuclease-free water.

[0011] A method for detecting echinococcosis pathogens for non-diagnostic and non-therapeutic purposes, comprising the following steps: (1) Extract DNA from the sample to be tested; (2) Perform PCR amplification using the above primer combination or kit, and determine whether the sample contains the corresponding echinococcosis pathogen based on the amplification results.

[0012] Furthermore, the PCR reaction system includes 12.5 μL of DNA polymerase mixture, 1 μL of DNA template with a concentration greater than 50 ng / μL, 0.75 μL (10 μM) of specific primers F1 / R1, 1.5 μL (10 μM) of specific primers F2 / R2 and 0.35 μL (10 μM) of specific primers F3 / R3, and finally, double-distilled water to make up to 25 μL.

[0013] Furthermore, the PCR reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s; 56℃ annealing for 40 s; 72℃ extension for 40 s; a total of 35 cycles; 72℃ extension for 5 min; and storage at 4℃ after the reaction was completed.

[0014] Furthermore, if the length of the amplified target fragment is 556bp, 234bp, and / or 406bp, it indicates that the sample contains Echinococcus multilocularis, Echinococcus granulosus, and / or Echinococcus shiquensis.

[0015] The beneficial effects of this invention are: This invention establishes a multiplex PCR method for identifying Echinococcus granulosus, Echinococcus multilocularis, and Echinococcus shiquensis in feces and tissues. This method is highly specific and sensitive, and has the advantages of high throughput, simplicity, and speed. It can significantly improve work efficiency and reduce time and economic costs, and has broad application prospects in the field of molecular epidemiological investigation of echinococcosis.

[0016] This invention utilizes primers designed based on the mitochondrial genes of *Echinococcus granulosus*, *Echinococcus multilocularis*, and *Echinococcus shiquensis*. Primer design and screening are crucial for multiplex PCR, as their specificity and sensitivity directly determine the overall specificity and sensitivity of the method. Primer design for multiplex PCR is far more challenging than for singlex PCR because multiple primer pairs are involved in a single system, increasing the risk of cross-reactions with closely related pathogens, leading to false positives. Furthermore, complementary binding between primer pairs can easily occur, causing interference. Additionally, multiplex PCR requires a certain difference in amplified product length to distinguish bands via electrophoresis, further complicating primer design. The three primer pairs in this invention do not interfere with each other, exhibit high specificity, and do not cross-react with any of the five closely related tapeworms, or produce bands of different sizes due to cross-reactions, thus not interfering with result interpretation.

[0017] Furthermore, the primers designed in this invention exhibit high sensitivity, achieving detection limits of 0.0004 ng / μL for all three pathogen DNAs. In contrast, existing technologies only achieve sensitivities of 20 pg, 10 pg, and 20 pg for the same three pathogens. Therefore, the technical solution developed in this invention is significantly superior to currently known multiplex PCR techniques capable of simultaneously detecting these three pathogens. Using these specific primers and following the method of this invention, multiplex PCR detection of samples can achieve simultaneous detection of three pathogens in humans, intermediate hosts, and definitive hosts in my country: *Echinococcus granulosus*, *Echinococcus multilocularis*, and *Echinococcus shiquensis*. Compared to previous methods, this significantly reduces detection costs and time, improves detection efficiency and accuracy, and is suitable for rapid and accurate pathogen identification in clinical and laboratory settings. Attached Figure Description

[0018] Figure 1 Electrophoresis images of PCR results obtained by using three pairs of specific detection primers to specifically amplify DNA templates of three target echinococcosis pathogens, both individually and in combination. Lane 1 is the negative control; Lane 2 shows the DNA molecular weight standard marker II; Lane 3 shows the PCR results of amplifying mixed DNA templates of *Echinococcus granulosus*, *Echinococcus multilocularis*, and *Echinococcus shiquensis* using three mixed primers; Lanes 4-6 show the PCR results of amplifying DNA templates of *Echinococcus multilocularis*, *Echinococcus granulosus*, and *Echinococcus shiquensis* using three mixed primers respectively; Lane 7 shows the PCR results of amplifying mixed templates of *Echinococcus granulosus* and *Echinococcus multilocularis* using three mixed primers; Lane 8 shows the PCR results of amplifying mixed DNA templates of *Echinococcus granulosus* and *Echinococcus shiquensis* using three mixed primers; Lane 9 shows the PCR results of amplifying mixed DNA templates of *Echinococcus multilocularis* and *Echinococcus shiquensis* using three mixed primers. Figure 2 Electrophoresis results of PCR using three pairs of specific detection primers to specifically detect DNA templates from three target echinococcosis pathogens and five closely related non-target echinococcosis species. Lane 1 contains DNA molecular weight standard marker II; lanes 2, 6, and 9 contain PCR results of amplifying the target worm species Echinococcus granulosus, Echinococcus multilocularis, and Echinococcus shiquensis using mixed primers, respectively; the other lanes, from left to right, contain PCR results of amplifying Taenia vesicularis, Taenia macrophylla, Taenia asiaticus, Taenia bovis, and Taenia solium. Figure 3 Electrophoresis results of PCR using three pairs of primers to detect the sensitivity of three different concentrations of DNA templates from three target echinococcosis pathogens; Lane 1 contains the standard amount of DNA molecules, while lanes 2-14 contain the target insect DNA as template. The final template concentrations in the reaction system were 0.04 ng / μL, 0.02 ng / μL, 0.01 ng / μL, 0.004 ng / μL, 0.002 ng / μL, 0.001 ng / μL, 0.0004 ng / μL, 0.0002 ng / μL, 0.0001 ng / μL, 0.00004 ng / μL, 0.00002 ng / μL, 0.00001 ng / μL, and 0.000004 ng / μL, respectively. Figure 4 Electrophoresis results of PCR using three pairs of primers to detect the sensitivity of three different concentrations of DNA templates from three target echinococcosis pathogens; Lane 1 contains the standard amount of DNA molecules, while lanes 2-14 contain the target insect DNA as template. The final template concentrations in the reaction system were 0.04 ng / μL, 0.02 ng / μL, 0.01 ng / μL, 0.004 ng / μL, 0.002 ng / μL, 0.001 ng / μL, 0.0004 ng / μL, 0.0002 ng / μL, 0.0001 ng / μL, 0.00004 ng / μL, 0.00002 ng / μL, 0.00001 ng / μL, and 0.000004 ng / μL, respectively. Figure 5 Electrophoresis results of PCR using three pairs of primers to detect the sensitivity of three different concentrations of DNA templates from three target echinococcosis pathogens; Lane 1 contains the standard amount of DNA molecules, while lanes 2-14 contain the target insect DNA as template. The final template concentrations in the reaction system were 0.04 ng / μL, 0.02 ng / μL, 0.01 ng / μL, 0.004 ng / μL, 0.002 ng / μL, 0.001 ng / μL, 0.0004 ng / μL, 0.0002 ng / μL, 0.0001 ng / μL, 0.00004 ng / μL, 0.00002 ng / μL, 0.00001 ng / μL, and 0.000004 ng / μL, respectively. Figure 6 Electrophoresis results of PCR performed using fecal samples from canines, human echinococcosis lesions, and intermediate host lesions as DNA templates; In this study, lane 1 represents the DNA molecular weight standard marker II; lanes 2-4 represent the amplification results of patient lesions, which were infected with Echinococcus granulosus, Echinococcus multilocularis, and Echinococcus multilocularis, respectively; lanes 5-6 represent the amplification results of intermediate host lesions, which were infected with Echinococcus shiquensis and Echinococcus granulosus, respectively; lane 7 represents the amplification results of fecal samples from dogs infected with Echinococcus granulosus; lane 8 is the negative control; and lane 9 is the blank control. Detailed Implementation

[0019] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0020] In the following examples, unless otherwise specified, all experimental conditions were performed according to conventional conditions well known to those skilled in the art, such as those described in *Molecular Cloning: A Laboratory Manual* by Sambrook et al. (New York: Cold Spring Harbor Laboratory Press, 1989) and *The Laboratory Animal Standards Manual* (National Laboratory Animal Standards Center, November 2004): Basic Technical Guide, Fifth Edition (John Wiley & Sons, Inc., 2005), or according to the manufacturer's recommended conditions and procedures. Unless otherwise specified, all materials and reagents used in the examples were commercially available through conventional methods.

[0021] Example 1: Construction of primer pairs Primer pairs were designed to detect *Echinococcus multilocularis*, *Echinococcus granulosus*, and *Echinococcus shiquensis*, as follows: Echinococcus multilocularis Em-F1: 5'-GTCAGTCTTGTTGTTGTGTT-3'; (SEQ ID NO.1) Em-R1: 5'-TACTCTAGTAGCCTCAGCAT-3'; (SEQ ID NO. 2) Echinococcus granulosus in the narrow sense Eg-F2: 5'-AGGTCGGTTCGATGTGCTTT-3'; (SEQ ID NO.3) Eg-R2: 5'-CCCGCTGACCAACTCTCTTT-3'; (SEQ ID NO.4) Echinococcus shiquensis Es-F3: 5'-GAGAATGTTGTGATTAGGTAGG-3'; (SEQ ID NO.5) Es-R3: 5'-CGAAGAAGATAACGGAGGAT-3'. (SEQ ID NO.6) Example 2: Construction of Detection Method 1. Extraction of genomic DNA from the sample to be tested Take 15-25 mg of worm tissue and extract whole genome DNA from the sample according to the instructions of the tissue DNA extraction kit.

[0022] 2. PCR amplification system, conditions, and result observation: Prepare the PCR reaction solution in a 25 μL reaction tube. This includes: 12.5 μL DNA polymerase mixture, 1 μL DNA template with a concentration greater than 50 ng / μL (this concentration is used for specificity experiments, but the detection of the sample is not limited to this), 0.75 μL (10 μM) specific primers F1 / R1, 1.5 μL (10 μM) specific primers F2 / R2, and 0.35 μL (10 μM) specific primers F3 / R3, and double-distilled water to a final volume of 25 μL.

[0023] The PCR reaction tubes were placed in a PCR instrument for the reaction. The thermal cycling program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s; 56℃ annealing for 40 s; 72℃ extension for 40 s; for a total of 35 cycles; 72℃ extension for 5 min. After the reaction, the product was stored at 4℃. The PCR products were electrophoresed in 2% agarose gel (containing 0.5 μg / ml Golden View) at 80-110 V, and the results were observed using a UV gel imaging system.

[0024] The presence or absence of electrophoretic bands and the size of the bands determine whether it is a parasitic disease and the type of parasite that is infected. The target fragment lengths amplified by primer pair 1, primer pair 2 and primer pair 3 are 556bp, 234bp and 406bp, respectively.

[0025] Example 3 Specificity Detection The primer pairs designed in Example 1 and the detection method constructed in Example 2 were used to detect the echinococcosis pathogen, verifying the specificity of the primer pairs and the method. The specific experimental groups are as follows: (1) Specificity experiment of amplifying DNA templates of three target Echinococcus tapeworms using three pairs of mixed primers designed in this invention; (2) Specificity experiment of amplifying DNA mixed templates of three target Echinococcus tapeworms using the three pairs of mixed primers designed in this invention; (3) Specificity experiment of amplifying DNA mixed templates of two target Echinococcus tapeworms using three pairs of mixed primers designed in this invention; (4) Specificity experiments were conducted using the three pairs of mixed primers designed in this invention to amplify DNA templates from three target Echinococcus tapeworms and other closely related tapeworms. The other closely related tapeworms were: The following tapeworms were selected: *T. hydatigena*, *T. taeniaformis*, *T. asiatica*, *T. saginata*, and *T. soli* (μM). Whole-genome DNA was extracted according to the DNA extraction kit instructions. The extracted DNA was then subjected to PCR detection and sequencing. The sequences were compared with corresponding sequences in the NCBI gene bank to determine the species. The DNA samples after this identification served as DNA template standards.

[0026] Test results as follows Figure 1 and 2 As shown, the three primer pairs of this invention amplified specific single-target bands of 556bp, 234bp, and 406bp, respectively, corresponding to Echinococcus multilocularis, Echinococcus granulosus, and Echinococcus shiquensis. The amplified fragment sizes were consistent with expectations, indicating that this method can specifically detect the three target Echinococcus species. The three primer pairs do not interfere with each other when mixed, and can amplify only the target band of the corresponding species.

[0027] Moreover, this invention can only detect the three target insect species, and has no cross-reaction with the other five closely related insect species, or the size of the band formed by the cross-reaction is a non-target band and does not affect the judgment of the detection results. Therefore, it has strong specificity.

[0028] Example 4 Accuracy Verification The PCR products of *Echinococcus multilocularis*, *Echinococcus granulosus*, and *Echinococcus shiquensis* from Example 1 were sent to a biotechnology company for bidirectional sequencing. The sequence results were uploaded to NCBI for BLAST comparison, confirming the corresponding species.

[0029] Example 5 Sensitivity Detection Three target parasite DNAs were used as templates, with final template concentrations of 0.04 ng / μL, 0.02 ng / μL, 0.01 ng / μL, 0.004 ng / μL, 0.002 ng / μL, 0.001 ng / μL, 0.0004 ng / μL, 0.0002 ng / μL, 0.0001 ng / μL, 0.00004 ng / μL, 0.00002 ng / μL, 0.00001 ng / μL, and 0.000004 ng / μL. Amplification was performed using the primer pairs designed in Example 1 and the detection method constructed in Example 2. The results are shown in [Figure 1]. Figures 3-5 .

[0030] like Figures 3-5As shown, lane 1 represents the DNA molecular standard, and lanes 2-14 contain the target worm DNA as template. The final template concentrations in the reaction system were 0.04 ng / μL, 0.02 ng / μL, 0.01 ng / μL, 0.004 ng / μL, 0.002 ng / μL, 0.001 ng / μL, 0.0004 ng / μL, 0.0002 ng / μL, 0.0001 ng / μL, 0.00004 ng / μL, 0.00002 ng / μL, 0.00001 ng / μL, and 0.000004 ng / μL. The limits of detection for *Echinococcus multilocularis* DNA and *Echinococcus granulosus* DNA were both 0.0004 ng / μL, and the limit of detection for *Echinococcus shiquensis* DNA was 0.00004 ng / μL. As can be seen, the detection limits of this invention for the DNA of the three target parasites are all higher than 0.0004 ng / μL, indicating that it has good sensitivity.

[0031] Example 6: Verification using actual samples DNA was collected from three patients, two intermediate host lesions, and one canine fecal sample. The infecting parasite species was identified by sequencing. These six identified DNA samples were then used as templates for multiplex PCR amplification. The results are shown below. Figure 6 .

[0032] like Figure 6 As shown, lane 1 represents the DNA molecular weight standard marker II; lanes 2-4 represent amplification results from patient lesions infected with *Echinococcus granulosus*, *Echinococcus multilocularis*, and *Echinococcus multilocularis*, respectively; lanes 5-6 represent amplification results from intermediate host lesions infected with *Echinococcus shiquensis* and *Echinococcus granulosus*, respectively; lane 7 represents amplification results from fecal samples from dogs infected with *Echinococcus granulosus*; lane 8 is the negative control; and lane 9 is the blank control. Sequencing results were consistent with amplification results.

[0033] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A primer combination for simultaneously detecting three echinococcosis pathogens, characterized in that, The primer combination includes the following primer sequences: Em-F1: 5'-GTCAGTCTTGTTGTTGTGTT-3'; Em-R1: 5'-TACTCTAGTAGCCTCAGCAT-3'; Eg-F2: 5'-AGGTCGGTTCGATGTGCTTT-3'; Eg-R2: 5'-CCCGCTGACCAACTCTCTTT-3'; Es-F3: 5'-GAGAATGTTGTGATTAGGTAGG-3'; Es-R3: 5'-CGAAGAAGATAACGGAGGAT-3'.

2. A kit for simultaneously detecting three echinococcosis pathogens, characterized in that, Includes the primer combination described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, 2×Taq PCR amplification reagent with gel loading dye, and nuclease-free water.

4. A method for detecting echinococcosis pathogens for non-diagnostic and non-therapeutic purposes, characterized in that, Includes the following steps: (1) Extract DNA from the sample to be tested; (2) Perform PCR amplification using the primer combination described in claim 1, or the kit described in claim 2 or 3, and determine whether the sample contains the corresponding echinococcosis pathogen based on the amplification results.

5. The detection method according to claim 4, characterized in that, The PCR reaction system includes 12.5 μL of DNA polymerase mixture, 1 μL of DNA template with a concentration greater than 50 ng / μL, 0.75 μL (10 μM) of specific primers F1 / R1, 1.5 μL (10 μM) of specific primers F2 / R2 and 0.35 μL (10 μM) of specific primers F3 / R3, and finally, double-distilled water to make up to 25 μL.

6. The detection method according to claim 4, characterized in that, The PCR reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s; 56℃ annealing for 40 s; 72℃ extension for 40 s; a total of 35 cycles; 72℃ extension for 5 min; and storage at 4℃ after the reaction was completed.

7. The detection method according to claim 4, characterized in that, If the length of the amplified target fragment is 556bp, 234bp, and / or 406bp, it indicates that the sample contains Echinococcus multilocularis, Echinococcus granulosus, and / or Echinococcus shiquensis.

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