Composition and kit for detecting echinococcosis typing and / or prognosis and application

Abstract

The invention belongs to the field of molecular biology detection, specifically relates to detection of echinococcosis, and more specifically relates to detection of echinococcosis typing and / or prognosis. The composition provided by the invention has the advantages of higher detection sensitivity (up to 500 copies / mL), good specificity and more accurate detection, provides more sufficient and rapid diagnosis for clinicians, shortens the time of the clinicians for diagnosing the condition of a patient, and accelerates the implementation of treatment measures on the patient.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, specifically, it relates to the detection of echinococcosis, and more specifically, it relates to the AE / CE typing of echinococcosis and the detection of the prognosis of echinococcosis. Background Technology

[0002] Echinococcosis, also known as echinococcosis, is a serious zoonotic parasitic disease caused by infection with the larvae of Echinococcus spp., which seriously endangers human and animal health. Among the most common species are Echinococcus granulosus and Echinococcus multilocularis, which can cause cystic echinococcosis (CE) and alveolar echinococcosis (AE), respectively, severely impacting the health of residents in endemic areas and hindering socioeconomic development. Echinococcosis has an insidious onset in humans, a relatively long course, and complex clinical manifestations, often without obvious symptoms or signs in the early stages. Currently, there is a lack of effective and sensitive methods for diagnosing echinococcosis, relying mainly on imaging diagnosis (ultrasound, MRI, CT). However, patients diagnosed by imaging often already have substantial lesions in organs such as the liver, causing them to miss the optimal treatment window. Although there are currently molecular biological diagnostic methods based on serum antibodies, the antigenic substances differ at different developmental stages of the Echinococcus larvae's life cycle, and its proteins have cross-antigens with tapeworms and other pathogenic microorganisms, resulting in a high false positive rate in immunodiagnostic results. Therefore, these methods can only be used as auxiliary detection indicators.

[0003] MicroRNAs (miRNAs) are a class of small, non-coding RNAs with lengths of 19–25 nucleotides. They primarily participate in regulating life activities such as individual development, apoptosis, proliferation, and differentiation by completely or incompletely pairing with the 3'UTR of target genes, degrading the mRNA of target genes, or inhibiting their translation. Secretory miRNAs, especially those secreted by extracellular vesicles (EVs) such as exosomes, may mediate paracrine and endocrine communication between different tissues, thereby regulating gene expression and remotely controlling cellular function. In parasitic infections, EVs secreted by parasites contain a large number of parasite-derived miRNAs. These parasite-derived miRNAs have conserved mechanisms of action, stable physicochemical properties, and can be taken up by nearby host immune or tissue cells. They play an important role in parasitic maintenance and pathogenicity through cross-species regulation.

[0004] Furthermore, after infection with pathogens, multiple host miRNAs participate in the regulation of immune network responses to stimuli and pathogens, forming a complex regulatory network. Therefore, miRNAs can serve as biomarkers for monitoring the occurrence, development, diagnosis, and prognosis of parasitic diseases. Compared with existing imaging and tissue biopsies, blood testing offers unique advantages such as convenient sampling, low cost of early screening, easy sample collection, and low background interference.

[0005] However, current miRNA detection technologies have certain shortcomings in terms of sensitivity and specificity, as well as ease and speed of operation. For example, the current tailing method can only distinguish between miRNAs with significant sequence differences; the current TaqMan method can only detect one miRNA target per tube per experiment; and all of the above methods require separate reverse transcription and amplification experiments, which are prone to environmental and cross-contamination during the tube opening process, making experimental operation and the elimination of abnormal results more complex. Therefore, in this invention, by introducing a pair of long and short universal primer sequences and optimizing the reaction procedure, the existing miRNA detection technology is improved, the reaction time is shortened, the two-step method is combined into a one-step detection, and three to five miRNA targets can be detected simultaneously in one tube.

[0006] Therefore, there is a need in this field for a product that can easily and quickly detect echinococcosis with high sensitivity and specificity, so as to provide clinicians with a more comprehensive rapid diagnosis and suggestions for different treatment measures. Summary of the Invention

[0007] In view of this, in a first aspect, the present invention provides a composition for detecting echinococcosis typing and / or prognosis, comprising:

[0008] The first nucleic acid composition comprises the primer and probe set shown below:

[0009] The upstream primer, downstream primer, and probe for detecting hsa-miR125b-5p, as shown in SEQ ID NO:1–3; and / or

[0010] The upstream primer, downstream primer, and probe for detecting emu-miR10-5p are shown in SEQ ID NO:4-6;

[0011] The second nucleic acid composition comprises the primer and probe set shown below:

[0012] The upstream primer, downstream primer, and probe for detecting emu-miR277-5p, as shown in SEQ ID NO:7–9; and / or

[0013] The upstream primer, downstream primer, and probe for detecting egr-miR71-5p, as shown in SEQ ID NO:10-12; and

[0014] The third nucleic acid composition includes the primer and probe set shown below:

[0015] The upstream primer, downstream primer, and probe for detecting hsa-miR155-5p, as shown in SEQ ID NO:13-15; and / or

[0016] The upstream primer, downstream primer, and probe for detecting hsa-let7a-5p are shown in SEQ ID NO:16-18.

[0017] Furthermore, the present invention provides a composition for detecting echinococcosis typing and / or prognosis, comprising:

[0018] The first nucleic acid composition comprises the primer and probe set shown below:

[0019] The upstream primer, downstream primer, and probe for detecting hsa-miR125b-5p are shown in SEQ ID NO:1-3;

[0020] The upstream primer, downstream primer, and probe for detecting emu-miR10-5p are shown in SEQ ID NO:4-6;

[0021] The second nucleic acid composition comprises the primer and probe set shown below:

[0022] The upstream primer, downstream primer, and probe for detecting emu-miR277-5p are shown in SEQ ID NO:7-9;

[0023] The upstream primer, downstream primer, and probe for detecting egr-miR71-5p, as shown in SEQ ID NO:10-12; and

[0024] The third nucleic acid composition includes the primer and probe set shown below:

[0025] The upstream primer, downstream primer, and probe for detecting hsa-miR155-5p are shown in SEQ ID NO:13-15.

[0026] The upstream primer, downstream primer, and probe for detecting hsa-let7a-5p are shown in SEQ ID NO:16-18.

[0027] Furthermore, the composition further includes:

[0028] The fourth nucleic acid composition comprises the primer and probe set shown below:

[0029] The upstream primer, downstream primer, and probe for detecting egr-miR2a-3p, as shown in SEQ ID NO:19–21; and / or

[0030] The upstream primer, downstream primer, and probe for detecting egr-let7-5p are shown in SEQ ID NO:22-24.

[0031] The combined detection composition provided by this invention mainly utilizes multiplex fluorescent PCR analysis to simultaneously detect echinococcosis AE / CE typing and prognosis in a single-tube reaction system, providing targeted strategies for subsequent treatment. The composition of this invention offers higher detection sensitivity (up to 500 copies / mL), better specificity, and more accurate detection, providing clinicians with a more comprehensive and rapid diagnosis, shortening the time required for patient diagnosis, and accelerating the implementation of treatment measures.

[0032] Furthermore, when the first nucleic acid composition tests positive, the sample is infected with Echinococcus multilocularis.

[0033] Furthermore, when the third nucleic acid composition tests positive, the sample is infected with Echinococcus granulosus.

[0034] Furthermore, when the tests for the first, second, and third nucleic acid combinations are all positive, the sample indicates a co-infection with Echinococcus multilocularis and Echinococcus granulosus.

[0035] Furthermore, if the Ct value of the fourth nucleic acid composition sample detected before surgery for echinococcosis is less than the Ct value detected after surgery for echinococcosis, then the prognosis of the echinococcosis in the sample is good.

[0036] The term "positive test" means that the fluorescent channel corresponding to the probe in each nucleic acid composition has a clear S-shaped amplification curve and a Ct value ≤ 40.

[0037] Furthermore, the fluorescent groups of the probes in each nucleic acid composition of the present invention are different from each other and do not interfere with each other.

[0038] Furthermore, the probes inside each nucleic acid composition may be the same or different.

[0039] In this article, "dissimilar and non-interfering" means that each probe in the composition uses a different fluorophore and will not affect the detection of each other, i.e., different channels can be used for detection. For example, ATTO425, Quasar705, FAM, HEX, ROX, and CY5 can be used. These groups have different absorbance values ​​and can be selected in different channels, thus avoiding mutual interference.

[0040] In some specific implementations, the fluorescent reporter group of the probes hsa-miR125b-5p and emu-miR10-5p is FAM; the fluorescent reporter group of the probes emu-miR277-5p and egr-miR71-5p is HEX; the fluorescent reporter group of the probes hsa-miR155-5p and hsa-let7a-5p is ROX; and the fluorescent reporter group of the probes egr-miR2a-3p and egr-let7-5p is Quasar705.

[0041] In some specific embodiments, the composition further includes a fifth nucleic acid composition, comprising an upstream primer, a downstream primer, and a probe for detecting an internal standard.

[0042] In one specific embodiment, the composition further includes a fifth nucleic acid composition comprising upstream primers, downstream primers, and probes for detecting internal standards as shown in SEQ ID NO: 25–27.

[0043] Furthermore, in some embodiments, the composition of the present invention may simultaneously comprise one or more sets of the primer and probe pairs described above. In the present invention, a "set" refers to mutually matching upstream and downstream primers and probes for detecting a target.

[0044] The compositions of this invention can be arbitrarily combined to detect any combination of eight corresponding targets. Those skilled in the art can combine them as needed, determining which targets to detect by combining the primer and probe pairs corresponding to those targets. All such combinations are included in this invention.

[0045] For example, it may include any 7 sets of the above 8 sets of primers and probes, any 6 sets of the above 8 sets of primers and probes, any 5 sets of the above 8 sets of primers and probes, any 4 sets of the above 8 sets of primers and probes, any 3 sets of the above 8 sets of primers and probes, any 2 sets of the above 8 sets of primers and probes, or any 1 set of the above 8 sets of primers and probes.

[0046] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0047] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0048] Furthermore, the 3' end of the probe also has a quenching group, such as BHQ1 or BHQ2.

[0049] In one specific implementation, the 3' end of the probe is BHQ1.

[0050] In one specific embodiment, each component of the composition of the present invention is contained in a separate package.

[0051] In one specific embodiment, the components of the composition of the present invention are contained in the same package.

[0052] Furthermore, the components of the composition of the present invention exist in a mixed form.

[0053] Secondly, the present invention provides the use of the above-described composition of the present invention in the preparation of a kit for detecting echinococcosis typing and / or prognosis.

[0054] Thirdly, the present invention provides a kit for detecting echinococcosis typing and / or prognosis, the kit comprising the composition of the present invention as described above.

[0055] Furthermore, the kit also includes negative and positive controls.

[0056] In one specific implementation, the negative control is at least one of DEPC H2O and physiological saline. The positive control is at least one of the plasmids or gene fragments of the corresponding target, such as HSA-MIR125B-5P, EMU-MIR10-5P, EMU-MIR277-5P, EGR-MIR71-5P, and Hsa-miR155-5p.

[0057] Furthermore, the kit also includes dNTPs, PCR buffer, and Mg. 2+ At least one of them.

[0058] Furthermore, the kit also includes at least one of the following: nucleic acid release reagent, nucleic acid extraction reagent, and nucleic acid amplification reagent.

[0059] Furthermore, the kit also includes nucleic acid release reagents, nucleic acid extraction reagents, dNTPs, reverse transcriptase, DNA polymerase, PCR buffer, and Mg2+. 2+ At least one of them.

[0060] Furthermore, the concentration of the DNA polymerase is 3 U / reaction to 15 U / reaction, for example, the DNA polymerase can be Taq polymerase.

[0061] In one specific embodiment, the kit of the present invention includes Taq enzyme, RT enzyme, and Mg 2+ dNTPs, primers, probes, and PCR buffer.

[0062] The PCR buffer components include Tris-HCl, KCl, and Triton X-100, with a total volume of 20 μl to 200 μl in a single PCR reaction tube.

[0063] Fourthly, a method for detecting echinococcosis typing for non-diagnostic purposes is provided, the method comprising the following steps:

[0064] 1) Extract or release nucleic acid from the sample to be tested;

[0065] 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;

[0066] 3) Obtain and analyze the results.

[0067] In this invention, the sample used for detection can be cyst fluid, plasma, urine, or feces, but is not limited to these.

[0068] In one specific embodiment, the use of a composition for preparing a reagent for detecting echinococcosis typing and prognosis is provided, the detection comprising the following steps:

[0069] 1) Extract nucleic acid from the sample to be tested;

[0070] 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition or kit of the present invention as described above;

[0071] 3) Obtain and analyze the results.

[0072] In this article, the term "non-diagnostic purpose" refers to something not intended to obtain information about whether an individual is infected with the aforementioned pathogens and suffers from illnesses such as food poisoning. For example, it may be necessary to test for the presence of the aforementioned pathogens in a test culture. Attached Figure Description

[0073] Figures 1-4 The images show the detection results of the composition of the present invention (samples of Echinococcus multilocularis, Echinococcus granulosus, Echinococcus multilocularis and Echinococcus granulosus, and postoperative samples of echinococcosis).

[0074] Figure 5 This is a graph showing the specificity of the composition of the present invention;

[0075] Figures 6-13 The images show the detection results of the compositions of the present invention and the comparative compositions (the detection results of hsa-miR125b-5p, emu-miR10-5p, emu-miR277-5p, egr-miR71-5p, hsa-miR155-5p, hsa-let7a-5p, egr-miR2a-3p, and egr-let7-5p, respectively). Detailed Implementation

[0076] The present invention will be described in detail below with reference to specific implementation schemes and embodiments, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific implementation schemes and embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0077] Example 1: Primers and probes used in this invention

[0078] The primers and probes used in this invention are shown in Table 1 below:

[0079] Table 1

[0080]

[0081]

[0082] The fluorescent reporter group for the probes hsa-miR125b-5p and emu-miR10-5p is FAM; the fluorescent reporter group for the probes emu-miR277-5p and egr-miR71-5p is HEX; the fluorescent reporter group for the probes hsa-miR155-5p and hsa-let7a-5p is ROX; and the fluorescent reporter group for the probes egr-miR2a-3p and egr-let7-5p is Quasar705.

[0083] Example 2: Method for detecting pathogens

[0084] Reagent preparation:

[0085] Based on the number of samples to be tested, positive controls, and negative controls, take the corresponding amounts of PCR reaction solution and enzyme mixture (35 μL PCR reaction solution / person + 10 μL enzyme mixture / person), mix thoroughly to form a PCR mixture, centrifuge at 2000 rpm for 10 seconds, and set aside for later use.

[0086] Preparation of enzyme mixture:

[0087] The enzyme mixture consists of a mixture of reverse transcriptase and Taq enzyme. It is prepared by mixing reverse transcriptase (5U) and Taq enzyme (15U) in a specific ratio.

[0088] Sample processing and loading

[0089] Take 300 μL of the test sample, negative control, and positive control into a 1.5 mL centrifuge tube, and perform nucleic acid extraction using the nucleic acid extraction or purification reagents from Sansure Biotech Inc. in accordance with their instructions.

[0090] Take 5 μL of the prepared sample, negative control and positive control and add them to the reaction tubes respectively. Add 45 μL of PCR mixture to each reaction tube and cap the tubes.

[0091] The real-time fluorescence PCR reaction system was prepared according to Table 2 below:

[0092] Table 2

[0093] reagents Dosage Final concentration PCR buffer 29.375μL 1X <![CDATA[1M Mg 2+ ]]> 0.8μL 16mM 100mM DNTP(U) 2μL 4mM 5U reverse transcriptase 5μL 1U / μL RNasin 0.125μL 10 U / μL 15U Taq enzyme 5μL 3U / μL Primers SEQ ID NO:1-9 (50μM) 0.1μL 0.1μM Primer SEQ ID NO:10-18 (500μM) 0.1μL 1μM Probe SEQ ID NO:19-27 (500μM) 0.1μL 1μM template 5μL /

[0094] PCR amplification was performed on PCR instruments such as the ABI 7500 Real-Time PCR System, the Yarui MA-6000 Real-Time PCR System, and the SLAN-96P Fully Automated Medical PCR Analysis System, following specific temperature and time settings. The PCR amplification program is shown in Table 3 below:

[0095] Table 3

[0096]

[0097] Results analysis:

[0098] In early screening for echinococcosis, if the FAM, HEX / VIC, Quasar, and CY5 channels show obvious S-shaped amplification curves and the Ct value is ≤40, it is considered positive for *Echinococcus multilocularis*. If the HEX / VIC, ROX, Quasar, and CY5 channels show obvious S-shaped amplification curves and the Ct value is ≤40, it is considered positive for *Echinococcus granulosus*. If the FAM, HEX / VIC, ROX, Quasar, and CY5 channels all show obvious S-shaped amplification curves and the Ct value is ≤40, it is considered positive for both *Echinococcus multilocularis* and *Echinococcus granulosus*. If the FAM, HEX / VIC, ROX, and Quasar channels show no amplification curve (NoCt) or the Ct value is >40, and the CY5 channel shows obvious S-shaped amplification curve, it is considered negative for echinococcosis.

[0099] In determining a positive result, there is no fixed requirement as to whether all channels show a line; see Table 4 for details.

[0100] Table 4

[0101]

[0102] Example 3: Detection results of test samples of the composition of the present invention

[0103] The primers and probes shown in Example 1 were used to detect samples of *Echinococcus multilocularis* and *Echinococcus granulosus* according to the method in Example 2. PCR detection was performed on a Hongshi real-time PCR instrument, and the results are as follows: Figures 1-4 As shown in the figure, the compositions of the present invention can detect and differentiate between Echinococcus multilocularis and Echinococcus granulosus, and can also detect prognosis.

[0104] Example 4: Sensitivity of the composition of the present invention

[0105] The LOD (sensitivity) of Echinococcus tapeworm samples was tested, and the results are shown in Table 5. The results show that the composition can still accurately detect samples with a sensitivity as low as 500 copies / mL, indicating that the sensitivity of the composition of the present invention is 500 copies / mL.

[0106] Table 5

[0107]

[0108] Example 5: Specificity of the composition of the present invention

[0109] The composition of this invention shows no cross-reactivity with other Echinococcus tapeworms (Echinococcus vortex, Echinococcus septemlobus, and Echinococcus shiquensis). Results are as follows... Figure 5 As shown, the compositions of the present invention have excellent specificity.

[0110] Example 6: Anti-interference properties of the composition of the present invention

[0111] The detection was performed according to the method described in Example 2, except that common interfering substances (fenbendazole (50 μg / mL), albendazole (50 μg / mL), heme (10 μg / mL), hemoglobin (10%)) were added to the entire system. The detection results are shown in Table 6. As can be seen from Table 6, the common interfering substances have no effect on the detection performance of the composition of the present invention.

[0112] Table 6

[0113]

[0114]

[0115] Comparative Example 1: Other primers and probes designed in this invention that do not perform well.

[0116] When using conventional one-step RNA amplification, non-specific amplification is amplified; and the content of circulating miRNA is very low, so one-step amplification will lose sensitivity. Therefore, the target involved in this invention was detected according to conventional RNA design methods. The process and time are shown in Table 7. The results show that the primers designed using the method of this invention do not require separate reverse transcription, and the total time is reduced by about one-third compared with the conventional tailing method.

[0117] The method of this invention refers to primers and probes designed according to the following method:

[0118] The upstream primer includes a first universal sequence and a first specific sequence, wherein the first specific sequence is complementary to 10-15 bases at the 5' end of the miRNA to be tested;

[0119] Downstream primers, wherein the downstream primers include a second universal sequence and a second specific sequence, wherein the second specific sequence is complementary to 6-10 bases at the 3' end of the first target miRNA; and

[0120] The probe comprises 4-8 bases at the 3' end of a first universal sequence and / or 4-8 bases at the 5' end of a second universal sequence, as well as a first miRNA sequence to be tested.

[0121] In the above method, the design of two specific sequences effectively improves detection sensitivity; the probe design can be flexibly adjusted within the designed region based on sequence differences between the target miRNAs, improving detection specificity; integrating the two-step tailing method into a one-step tailing method simplifies the operation steps and saves detection time; it enables the simultaneous detection of multiple mature miRNAs in one tube with only one PCR test, shortening detection time and improving detection efficiency. A comparison of PCR results with primers designed using conventional methods is shown below. Figures 6-13 As shown in the figure, the amplification effect of the primers and probes designed by this invention has a higher Ct value than that of primers and probes designed by the conventional tailing method. This indicates that the primers and probes designed by this invention have better amplification effect and higher sensitivity (both are multiplex PCR, and the results are presented for different targets).

[0122] Table 7

[0123] Detection methods Should reverse transcription be performed alone? Total time Adding tails yes Reverse transcription 30 minutes, PCR 90 minutes This invention no One-step method, 96 minutes

Claims

1. A composition for detecting a type and / or prognosis of echinococcosis, comprising: a first nucleic acid composition comprising a primer probe set as shown below: an upstream primer, a downstream primer and a probe for detecting hsa-miR125b-5p as shown in SEQ ID NO: 1-3; and / or an upstream primer, a downstream primer and a probe for detecting emu-miR10-5p as shown in SEQ ID NO: 4-6; a second nucleic acid composition comprising a primer probe set as shown below: an upstream primer, a downstream primer and a probe for detecting emu-miR277-5p as shown in SEQ ID NO: 7-9; and / or an upstream primer, a downstream primer and a probe for detecting egr-miR71-5p as shown in SEQ ID NO: 10-12; and a third nucleic acid composition comprising a primer probe set as shown below: an upstream primer, a downstream primer and a probe for detecting hsa-miR155-5p as shown in SEQ ID NO: 13-15; and / or an upstream primer, a downstream primer and a probe for detecting hsa-let7a-5p as shown in SEQ ID NO: 16-18.

2. The composition of claim 1, wherein, The composition comprises: a first nucleic acid composition comprising a primer probe set as shown below: an upstream primer, a downstream primer and a probe for detecting hsa-miR125b-5p as shown in SEQ ID NO: 1-3; an upstream primer, a downstream primer and a probe for detecting emu-miR10-5p as shown in SEQ ID NO: 4-6; a second nucleic acid composition comprising a primer probe set as shown below: an upstream primer, a downstream primer and a probe for detecting emu-miR277-5p as shown in SEQ ID NO: 7-9; an upstream primer, a downstream primer and a probe for detecting egr-miR71-5p as shown in SEQ ID NO: 10-12; and a third nucleic acid composition comprising a primer probe set as shown below: an upstream primer, a downstream primer and a probe for detecting hsa-miR155-5p as shown in SEQ ID NO: 13-15; an upstream primer, a downstream primer and a probe for detecting hsa-let7a-5p as shown in SEQ ID NO: 16-18.

3. The composition of claim 1, wherein, The composition further comprises: a fourth nucleic acid composition comprising at least one of the primer probe sets as shown below: an upstream primer, a downstream primer and a probe for detecting egr-miR2a-3p as shown in SEQ ID NO: 19-21; or an upstream primer, a downstream primer and a probe for detecting egr-let7-5p as shown in SEQ ID NO: 22-24.

4. The composition of claim 1, wherein, The composition further comprises: a fifth nucleic acid composition comprising a primer probe set as shown below: an upstream primer, a downstream primer and a probe for detecting an internal standard as shown in SEQ ID NO: 25-27.

5. The composition according to any one of claims 1 to 4, characterized in that, When the first nucleic acid composition is positive, the sample is infected with E. granulosus; when the third nucleic acid composition is positive, the sample is infected with E. multilocularis; when the first, second and third nucleic acid compositions are all positive, the sample is infected with both E. granulosus and E. multilocularis.

6. The composition of claim 5, wherein, The fluorescent reporter group of the probes for hsa-miR125b-5p and emu-miR10-5p is FAM; the fluorescent reporter group of the probes for emu-miR277-5p and egr-miR71-5p is HEX; the fluorescent reporter group of the probes for hsa-miR155-5p and hsa-let7a-5p is ROX; the fluorescent reporter group of the probes for egr-miR2a-3p and egr-let7-5p is Quasar705.

7. Use of the composition according to any one of claims 1-6 for the preparation of a kit for detecting the typing and / or prognosis of echinococcosis.

8. A kit for detecting the typing and / or prognosis of echinococcosis, comprising the composition according to any one of claims 1-6.

9. The kit according to claim 7 or 8, characterized in that, The kit further comprises at least one of the following: a nucleic acid releasing reagent, a nucleic acid extraction reagent, a nucleic acid amplification reagent.

10. Use of a composition for the preparation of a reagent for detecting the typing and / or prognosis of echinococcosis, the detection comprising the following steps: 1) extracting nucleic acid from the sample to be tested; 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition according to any one of claims 1-6 or the kit according to any one of claims 8-9; 3) obtaining and analyzing the results.

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