Composition for detecting and typing pathogens of echinococcosis, kit and application

Abstract

The invention belongs to the field of molecular biology detection, particularly relates to detection of echinococcosis, and more particularly relates to cfDNA markers and miRNA markers related to detection of echinococcosis. The composition disclosed by the invention is higher in detection sensitivity (up to 200 copies / mL), good in specificity and more accurate in detection, provides more sufficient and rapid diagnosis for clinicians, shortens the time for the clinicians to diagnose the condition of a patient, and accelerates the implementation of treatment measures on the patient.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to the detection of echinococcosis, and more specifically, to the detection and AE / CE typing of the pathogen of echinococcosis by detecting cfDNA and miRNA. Background Art

[0002] Echinococcosis, also known as echinococcosis, is a zoonotic parasitic disease caused by infection with the larvae of Echinococcus spp., which seriously endangers the health of humans and animals. With the application of phylogenetic analysis methods, Echinococcus is divided into 9 species that are currently recognized: Echinococcus granulosus, Echinococcus multilocularis, Echinococcus oligarthra, Echinococcus vogeli, Echinococcus shiquicus, Echinococcus felidis, Echinococcus equinus, Echinococcus ortleppi and Echinococcus canadensis. There is also a view that Echinococcus intermedius should be included. Among them, Echinococcus granulosus and Echinococcus multilocularis are the most common in China, which can cause cystic echinococcosis (CE) and alveolar echinococcosis (AE) respectively. The emergence of echinococcosis seriously affects the health of residents and social and economic development.

[0003] The onset of echinococcosis in the human body is insidious, the course of the disease is relatively long, the clinical manifestations are complex, and there are no obvious symptoms and signs in the early stage. At present, there is still a lack of effective and sensitive methods for the diagnosis of echinococcosis, which mainly relies on imaging diagnosis (B-ultrasound, MRI, CT), but patients diagnosed by imaging often have substantial lesions in organs such as the liver, causing patients to miss the best time for treatment. Although there are currently molecular biological diagnostic methods based on serum antibodies, due to the different antigenic substances in the life cycle of echinococcosis, and the presence of cross-antigens in its proteins and other pathogenic microorganisms such as tapeworms, the immune diagnosis results have a high false positive rate and can only be used as an auxiliary detection indicator.

[0004] Therefore, developing highly sensitive, specific, rapid and non-invasive methods and kits for early diagnosis of echinococcosis is a practical and effective means for early monitoring, screening, diagnosis and treatment of echinococcosis, and has broad application prospects.

[0005] Realtime fluorescence quantitative PCR (qPCR), with its advantages of high precision and quantification, is the mainstream technology platform for molecular diagnosis at this stage and the "gold standard" for clinical diagnosis. In the field of parasitic disease diagnosis, specific probes can be designed to detect the DNA of the target pathogenic microorganism. Currently, there is a method to determine whether the test animal is infected with echinococcosis by detecting the specific DNA of echinococcosis eggs in the feces of test animals such as dogs and foxes through PCR amplification. However, animals such as dogs are the final hosts of Echinococcus, with adult worms in their intestines and a large number of eggs in their feces. Humans are the intermediate hosts of Echinococcus, with no adult worms in their bodies and no eggs in their feces. Therefore, the above detection method is not suitable for humans. This field urgently needs a product that can simply and quickly detect echinococcosis with high sensitivity and good specificity, to provide clinicians with a more comprehensive rapid diagnosis and provide suggestions for different treatment measures. Summary of the invention

[0006] In view of this, in a first aspect, the present invention provides a composition for detecting and typing pathogens of echinococcosis, comprising:

[0007] The first nucleic acid composition comprises at least one of the following primer probe sets:

[0008] Upstream primers, downstream primers and probes for detecting AE typing as shown in SEQ ID NOs: 1, 2 and 3;

[0009] The upstream primer, downstream primer and probe for detecting CE typing as shown in SEQ ID NO: 1, 2 and 4; or

[0010] Universal detection upstream primers, downstream primers and probes as shown in SEQ ID NOs: 5, 6 and 7;

[0011] and a second nucleic acid composition comprising at least one of the primer probe sets shown below:

[0012] The upstream primer, downstream primer and probe for detecting emr-miR10-5p as shown in SEQ ID NO: 8, 9 and 10; or

[0013] The upstream primers, downstream primers and probe for detecting universal EG-let7-5p are shown in SEQ ID NOs: 11, 12 and 13.

[0014] The present invention designs a two-tube reaction system, extracts miRNA and cfDNA respectively, and then performs a qPCR test on the two-tube reaction systems under the same temperature program to complete the diagnosis and typing of echinococcosis, improves the existing detection technology, and increases the sensitivity and specificity of the detection.

[0015] The design of the present invention has the advantages of specificity, sensitivity, rapidity, simplicity, low invasiveness and low cost, and is conducive to solving the technical difficulties in the current early screening, diagnosis and epidemiological investigation of echinococcosis.

[0016] Further, the first nucleic acid composition includes at least two of the primer probe sets shown below:

[0017] Upstream primers, downstream primers and probes for detecting AE typing as shown in SEQ ID NOs: 1, 2 and 3;

[0018] The upstream primer, downstream primer and probe for detecting CE typing as shown in SEQ ID NO: 1, 2 and 4; or

[0019] The universal upstream primer, downstream primer and probe for detection are shown in SEQ ID NO: 5, 6 and 7.

[0020] Further, the first nucleic acid composition includes at least three of the primer probe sets shown below:

[0021] Upstream primers, downstream primers and probes for detecting AE typing as shown in SEQ ID NOs: 1, 2 and 3;

[0022] The upstream primer, downstream primer and probe for detecting CE typing as shown in SEQ ID NO: 1, 2 and 4; or

[0023] The universal upstream primer, downstream primer and probe for detection are shown in SEQ ID NO: 5, 6 and 7.

[0024] Further, the second nucleic acid composition includes at least two of the primer probe sets shown above.

[0025] The upstream primer, downstream primer and probe for detecting emr-miR10-5p as shown in SEQ ID NO: 8, 9 and 10; or

[0026] The upstream primers, downstream primers and probe for detecting universal EG-let7-5p are shown in SEQ ID NOs: 11, 12 and 13.

[0027] Furthermore, the present invention provides a composition for detecting and typing the pathogen of echinococcosis, comprising:

[0028] The first nucleic acid composition includes a primer probe set as shown below:

[0029] Upstream primers, downstream primers and probes for detecting AE typing as shown in SEQ ID NOs: 1, 2 and 3;

[0030] Upstream primers, downstream primers and probes for detecting CE typing as shown in SEQ ID NOs: 1, 2 and 4;

[0031] Universal detection upstream primers, downstream primers and probes as shown in SEQ ID NOs: 5, 6 and 7;

[0032] and a second nucleic acid composition comprising a primer probe set as shown below:

[0033] Upstream primers, downstream primers and probes for detecting emr-miR10-5p as shown in SEQ ID NOs: 8, 9 and 10;

[0034] The upstream primers, downstream primers and probe for detecting universal EG-let7-5p are shown in SEQ ID NOs: 11, 12 and 13.

[0035] Furthermore, the composition further comprises:

[0036] The third nucleic acid composition includes a primer probe set as shown below:

[0037] The upstream primer, downstream primer and probe for detecting the internal standard are shown in SEQ ID NOs: 14, 15 and 16.

[0038] Furthermore, the fluorescent groups of the probes in each nucleic acid composition of the composition of the present invention are different from each other and do not interfere with each other.

[0039] Furthermore, the probes in each nucleic acid composition may be the same or different.

[0040] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, FAM, HEX, ROX and CY5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other.

[0041] In some specific embodiments, the fluorescent reporter group of the probes for AE typing and emr-miR10-5p is FAM; the fluorescent reporter group of the probes for CE typing and EG-let7-5p is ROX, and the fluorescent reporter group of the universal probe is CY5.

[0042] Furthermore, for the diagnosis of cfDNA testing, when the FAM channel and CY5 channel tests are positive, the sample is infected with Echinococcus granulosus.

[0043] Furthermore, when the ROX channel and the CY5 channel are detected as positive, the sample is infected with Echinococcus multilocularis.

[0044] Furthermore, when the CY5 channel test is positive and the ROX channel and the FAM channel test are negative, the sample is infected with other species of Echinococcus.

[0045] Further, for the diagnosis of miRNA detection, when both the FAM channel and the ROX channel are positive or one of the channels is positive, the sample is infected with (multilocular and / or granular) Echinococcus.

[0046] The term "positive detection" means that the fluorescence channel corresponding to the probe in each nucleic acid composition has an obvious S-shaped amplification curve, and the Ct value is ≤40.

[0047] Further, in some embodiments, the composition of the present invention may include one or more of the above primer and probe pairs. In the present invention, "set" refers to upstream and downstream primers and probes that match each other for detecting a target.

[0048] The composition of the present invention can be combined into any combination of detection corresponding to 5 targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe pairs corresponding to the targets. These combinations are all included in the present invention.

[0049] For example, it may include any 4 of the above 5 sets of primers and probes, it may include any 3 of the above 5 sets of primers and probes, it may include any 2 of the above 5 sets of primers and probes, or it may include any 1 of the above 5 sets of primers and probes.

[0050] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0051] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0052] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

[0053] In a specific embodiment, the 3' end of the probe is BHQ1. In a specific embodiment, the first nucleic acid composition and the second nucleic acid composition are stored in different packages.

[0054] Furthermore, each component of the composition of the present invention is present in a separate package.

[0055] Furthermore, the components of each nucleic acid composition may be present in a mixed form.

[0056] In a second aspect, the present invention provides use of the above-mentioned composition of the present invention in preparing a kit for detecting and typing pathogens of echinococcosis.

[0057] In a third aspect, the present invention provides a kit for detecting and typing the pathogen of echinococcosis, the kit comprising the composition of the present invention as described above.

[0058] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0059] In a specific embodiment, the negative control is at least one of DEPC H2O and physiological saline. The positive control is a plasmid of pathogen cfDNA and a mixture of miRNA marker oligonucleotides.

[0060] Furthermore, the kit also includes dNTP, PCR buffer and Mg 2+ At least one of .

[0061] Furthermore, the kit also includes: at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, and a nucleic acid amplification reagent.

[0062] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, a reverse transcriptase, a DNA polymerase, a UNG enzyme, RNasin, a PCR buffer and Mg 2+ At least one of .

[0063] Furthermore, the concentration of the DNA polymerase is 3U / reaction to 15U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0064] In a specific embodiment, the kit of the present invention comprises UNG enzyme, RNasin, Taq enzyme, Mg 2+ , dNTPs, primers, probes and PCR buffer.

[0065] The components of the PCR buffer include Tris-HCl, KCl, and Triton X-100, and the total volume in a single PCR reaction tube is 20 μL to 200 μL.

[0066] In a fourth aspect, a method for detecting and typing the pathogen of echinococcosis for non-diagnostic purposes is provided, the method comprising the following steps:

[0067] 1) Extracting or releasing nucleic acid from the sample to be tested;

[0068] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0069] 3) Obtain and analyze the results.

[0070] In the present invention, the sample used for detection can be cystic fluid, plasma, urine or feces, etc., but is not limited thereto.

[0071] In a specific embodiment, a composition is provided for preparing a reagent for detecting and typing the pathogen of echinococcosis, wherein the detection comprises the following steps:

[0072] 1) Extracting nucleic acid from the sample to be tested;

[0073] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0074] 3) Obtain and analyze the results.

[0075] In this article, the term "non-diagnostic purpose" refers to not being intended to obtain information on whether an individual is infected with the above pathogens and suffers from diseases such as echinococcosis. For example, it may be required to detect whether the above pathogens are present in the test culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figures 1 to 3 Graphs showing the test results of the composition of the present invention (respectively, samples of Echinococcus multilocularis, samples of Echinococcus granulosus, and samples of Echinococcus multilocularis and Echinococcus granulosus).

[0077] Figure 4 It is a specific result diagram of the composition of the present invention.

[0078] Figure 5 This is a graph showing the detection results of comparative example 1 for cfDNA.

[0079] Figure 6 This is a graph showing the detection results of miRNA in Comparative Example 1. DETAILED DESCRIPTION

[0080] Cell free DNA (cfDNA) refers to highly fragmented DNA that exists in the human blood circulation and is free from cells. In certain circumstances (such as cancer patients, pregnant women, patients receiving organ transplants, etc.), a small amount of cfDNA from "heterogeneous" cells (such as tumor cells, fetal cells, or donor cells) can be used as a marker for genetic testing. The fragmented genomic DNA released after the rupture of dead tumor cells is also called ctDNA (circulating tumor DNA). The "liquid biopsy" technology based on cfDNA detection has received great attention in many aspects such as prenatal diagnosis, tumor screening, early diagnosis, treatment monitoring and prognosis evaluation.

[0081] MicroRNA (miRNA) is a type of non-coding small RNA with a length of 19-25 nucleotides. It mainly participates in regulating life activities such as individual development, cell apoptosis, proliferation and differentiation by completely or incompletely pairing with the 3'UTR of the target gene, degrading the target gene mRNA or inhibiting its translation. Secreted miRNAs, especially miRNAs secreted by extracellular vesicles (EVs) such as exosomes, may mediate paracrine and endocrine communications between different tissues, thereby regulating gene expression and remotely regulating cell functions. In parasitic infections, EVs secreted by the worm contain a large number of worm-derived miRNAs. These worm-derived miRNAs have a conservative mechanism of action, stable physical and chemical properties, can be taken up by nearby host immune or tissue cells, and play an important role in the maintenance of worm parasitism and pathogenicity through cross-species regulation.

[0082] In addition, after infection with pathogens, multiple host miRNAs participate in the regulatory immune network response to stimuli and pathogenic processes, and there is a complex regulatory network in the body. Therefore, miRNA can be used as a biomarker to monitor the occurrence, development, diagnosis and prognosis of parasitic diseases. Compared with existing imaging and tissue biopsies, blood tests have unique advantages such as convenient sampling, low cost for early screening, easy sample collection, and low background interference.

[0083] The present invention designs a two-tube reaction system, extracts miRNA and cfDNA respectively, and then performs qPCR detection on the two-tube reaction systems under the same temperature program to complete the diagnosis and typing of echinococcosis, improves the existing detection technology, and increases the sensitivity and specificity of the detection.

[0084] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0085] Example 1: Primers and probes used in the present invention

[0086] The primers and probes used in the present invention are shown in Table 1 below:

[0087] Table 1

[0088]

[0089] Among them, the fluorescent reporter group of the probe for AE typing and universal emr-miR10-5p is FAM; the fluorescent reporter group of the internal standard probe is HEX or VIC; the fluorescent reporter group of the probe for CE typing and universal EG-let7-5p is ROX; and the fluorescent reporter group of the universal probe is CY5.

[0090] Example 2: Method for detecting pathogens

[0091] Reagent preparation:

[0092] According to the number of samples to be tested, positive controls, and negative controls, take the corresponding amount of PCR reaction solution and enzyme mixture (PCR reaction solution 25.875 μL / person + enzyme mixture 4.125 μL / person), mix them thoroughly to form a PCR mixture, centrifuge at 2000 rpm for 10 seconds and set aside.

[0093] Preparation of enzyme mixture:

[0094] The enzyme mixture is composed of a reverse transcriptase and a Taq enzyme mixture, and is prepared by mixing reverse transcriptase (5U) and Taq enzyme (5U) in a certain ratio.

[0095] Sample handling and loading

[0096] Take 1000 μL of the sample to be tested, negative control, and positive control into a 2 mL centrifuge tube, and use the nucleic acid extraction or purification reagent of Shengxiang Biotechnology Co., Ltd. according to its instructions to extract nucleic acid, and extract cfDNA and miRNA respectively.

[0097] 20 μL of the above-treated samples, negative controls and positive controls were pipetted and added to reaction tubes, and 30 μL of PCR mixture was added to each reaction tube, and the tubes were capped.

[0098] The real-time fluorescence PCR reaction system for detecting cfDNA is configured according to Table 2 below:

[0099] Table 2

[0100] Reagents Dosage Final concentration PCR buffer I 20.975μL 1X <![CDATA[1M Mg 2+ ]]> 0.4μL 16mM 100mM DNTP(U) 1μL 4mM 5U reverse transcriptase 2μL 1U / μL UNG enzyme 0.1uL 1U / μL RNasin 0.125μL 10U / μL 5U Taq enzyme 1.9μL 3U / μL Primer SEQ ID NO: 1-2 (100 μM) 0.3μL 0.3μM Primer SEQ ID NO: 5-6 (100 μM) 0.6μL 0.6μM Primer SEQ ID NO: 8-9, 11-12 (100 μM) 0.2μL 0.2μM Primer SEQ ID NO: 14-15 (100 μM) 0.1μL 0.1μM Probe SEQ ID NO: 3 (100 μM) 0.05μL 0.05μM Probe SEQ ID NO:4 (100 μM) 0.15μL 0.15μM Probe SEQ ID NO:7 (100 μM) 0.3μL 0.3μM Probe SEQ ID NO: 10, 13, 16 (100 μM) 0.1μL 0.1μM template 20μL /

[0101] The real-time fluorescence PCR reaction system for detecting miRNA is configured according to the following Table 3:

[0102] Table 3

[0103] Reagents Dosage Final concentration PCR buffer II 20.975μL 1X <![CDATA[1M Mg 2+ ]]> 0.4μL 16mM 100mM DNTP(U) 1μL 4mM 5U reverse transcriptase 2μL 1U / μL UNG enzyme 0.1uL 1U / μL RNasin 0.125μL 10U / μL 5U Taq enzyme 1.9μL 3U / μL Primer SEQ ID NO: 1-2 (100 μM) 0.3μL 0.3μM Primer SEQ ID NO: 5-6 (100 μM) 0.6μL 0.6μM Primer SEQ ID NO: 8-9, 11-12 (100 μM) 0.2μL 0.2μM Primer SEQ ID NO: 14-15 (100 μM) 0.1μL 0.1μM Probe SEQ ID NO: 3 (100 μM) 0.05μL 0.05μM Probe SEQ ID NO:4 (100 μM) 0.15μL 0.15μM Probe SEQ ID NO:7 (100 μM) 0.3μL 0.3μM Probe SEQ ID NO: 10, 13, 16 (100 μM) 0.1μL 0.1μM template 20μL /

[0104] On PCR instruments such as ABI 7500 fluorescence quantitative PCR instrument, Yarui MA-6000 real-time quantitative PCR system, SLAN-96P fully automatic medical PCR analysis system, PCR amplification is performed according to certain temperature and time settings. The PCR amplification program is set as shown in Table 4 below:

[0105] Table 4

[0106]

[0107] Result analysis:

[0108] In the early screening of echinococcosis, for cfDNA detection, if the ROX and CY5 channels have obvious S-shaped amplification curves and the Ct value is ≤40, it is judged as positive for Echinococcus multilocularis; if the FAM and CY5 channels have obvious S-shaped amplification curves and the Ct value is ≤40, it is judged as positive for Echinococcus granulosus; if the CY5 channel has an obvious S-shaped amplification curve, but the FAM and ROX channels have no amplification curves, and the Ct value is ≤40, it is judged as positive for other species of Echinococcus; if the FAM, HEX / VIC, ROX, CY5 channels have no amplification curve (NoCt) or the Ct value is greater than 40, it is judged as negative for echinococcosis.

[0109] In the early screening of echinococcosis, for miRNA detection, if FAM or (and) ROX has an obvious S-shaped amplification curve and the Ct value is ≤40, it is judged to be positive for multilocular or granulosus Echinococcus; if the FAM, HEX / VIC, and ROX channels have no amplification curve (NoCt) or the Ct value is greater than 40, it is judged to be negative for echinococcosis.

[0110] In the determination of cfDNA positivity, there is no fixed requirement for whether all channels are started, as shown in Table 5.

[0111] Table 5

[0112]

[0113] In determining whether miRNA is positive, there is no fixed requirement for whether all channels are positive, as shown in Table 6.

[0114] Table 6

[0115]

[0116]

[0117] Example 3: Test results of the test samples of the composition of the present invention

[0118] The primers and probes shown in Example 1 were used to detect the samples of Echinococcus multilocularis and Echinococcus granulosus according to the method of Example 2. PCR detection was performed on a Macrostone fluorescent quantitative PCR instrument. The detection results are as follows: Figures 1-2 As shown, Figure 1 This is a multilocular Echinococcus sample after NGS sequencing for cfDNA detection. The FAM channel is NoCt (no line), and the ROX channel and CY5 channel are both lines, which is determined to be a multilocular Echinococcus sample; Figure 2This is a sample of Echinococcus granulosus after NGS sequencing for cfDNA detection. The ROX channel is NoCt (no line), and the FAM channel and CY5 channel are both lines, which is determined to be an Echinococcus granulosus sample; Figure 3 This is a sample of Echinococcus for miRNA detection, in which both the FAM channel and the ROX channel are positive, judging the presence of Echinococcus multilocularis or Echinococcus granulosus. It can be seen from the above figures that the composition of the present invention can detect and distinguish Echinococcus multilocularis from Echinococcus granulosus.

[0119] Example 4: Sensitivity of the composition of the present invention

[0120] The LOD (sensitivity) test of Echinococcus granulosus was performed, and the results are shown in Table 7, which shows that the composition can still accurately detect samples as low as 200 copies / mL, indicating that the sensitivity of the composition of the present invention is 200 copies / mL.

[0121] Table 7

[0122]

[0123]

[0124] Example 5. Specificity of the composition of the present invention

[0125] The composition of the present invention has no cross reaction with other Echinococcus tapeworms (Echinococcus fowleri, Echinococcus schrenkii, Echinococcus leonina, Echinococcus equi, Echinococcus ostreatus). Figure 4 As shown, it is shown that the composition of the present invention has good specificity.

[0126] Example 6: Anti-interference performance of the composition of the present invention

[0127] The detection was carried out according to the method described in Example 2, except that common interfering substances (heme (10 μg / mL), hemoglobin (10%)) were added to the whole system. The detection results are shown in Tables 8 and 9. It can be seen from Tables 8 and 9 that common interfering substances have no effect on the detection performance of the composition of the present invention.

[0128] Table 8

[0129]

[0130] Table 9

[0131]

[0132]

[0133] Comparative Example 1: Testing using only cfDNA

[0134] The target involved in the present invention was detected according to the method of detecting only cfDNA, the system used is shown in Table 10, and the process and time are shown in Table 11. The detection results are shown in Figure 5 and Figure 6 As shown, Figure 5 For the test results of cfDNA, Figure 6 For the detection results of miRNA, Figure 5 and Figure 6 It can be seen from the results that compared with the detection of cfDNA, the Ct value of the same sample detected by miRNA is significantly advanced, indicating that the sensitivity of the same sample detected by miRNA is significantly improved, indicating that the present invention detects miRNA and cfDNA respectively to complete the diagnosis and typing of echinococcosis, and has higher detection sensitivity and specificity.

[0135] Table 10

[0136] Reagents Dosage Final concentration PCR buffer I 20.975μL 1X <![CDATA[1M Mg 2+ ]]> 0.4μL 16mM 100mM DNTP(U) 1μL 4mM 5U reverse transcriptase 2μL 1U / μL UNG enzyme 0.1uL 1U / μL RNasin 0.125μL 10U / μL 5U Taq enzyme 1.9μL 3U / μL Primer SEQ ID NO: 1-2 (100 μM) 0.3μL 0.3μM Primer SEQ ID NO: 5-6 (100 μM) 0.6μL 0.6μM Primer SEQ ID NO: 8-9, 11-12 (100 μM) 0.2μL 0.2μM Primer SEQ ID NO: 14-15 (100 μM) 0.1μL 0.1μM Probe SEQ ID NO: 3 (100 μM) 0.05μL 0.05μM Probe SEQ ID NO:4 (100 μM) 0.15μL 0.15μM Probe SEQ ID NO:7 (100 μM) 0.3μL 0.3μM Probe SEQ ID NO: 10, 13, 16 (100 μM) 0.1μL 0.1μM template 20μL /

[0137] Table 11

[0138]

Claims

1. A composition for detecting and typing pathogens of echinococcosis, comprising: The first nucleic acid composition comprises at least one of the following primer probe sets: Upstream primers, downstream primers and probes for detecting AE typing as shown in SEQ ID NOs: 1, 2 and 3; The upstream primer, downstream primer and probe for detecting CE typing as shown in SEQ ID NO: 1, 2 and 4; or Universal detection upstream primers, downstream primers and probes as shown in SEQ ID NOs: 5, 6 and 7; and The second nucleic acid composition comprises at least one of the following primer probe sets: The upstream primer, downstream primer and probe for detecting emr-miR10-5p as shown in SEQ ID NO: 8, 9 and 10; or The upstream primers, downstream primers and probe for detecting universal EG-let7-5p are shown in SEQ ID NOs: 11, 12 and 13.

2. The composition according to claim 1, characterized in that The first nucleic acid composition includes at least two or at least three of the primer probe sets shown below: Upstream primers, downstream primers and probes for detecting AE typing as shown in SEQ ID NOs: 1, 2 and 3; The upstream primer, downstream primer and probe for detecting CE typing as shown in SEQ ID NO: 1, 2 and 4; or The universal upstream primer, downstream primer and probe for detection are shown in SEQ ID NO: 5, 6 and 7.

3. The composition according to claim 1, characterized in that The second nucleic acid composition comprises at least two of the primer probe sets shown below: The upstream primer, downstream primer and probe for detecting emr-miR10-5p as shown in SEQ ID NO: 8, 9 and 10; or The upstream primers, downstream primers and probe for detecting universal EG-let7-5p are shown in SEQ ID NOs: 11, 12 and 13.

4. The composition according to claim 1, characterized in that The composition comprises: The first nucleic acid composition includes a primer probe set as shown below: Upstream primers, downstream primers and probes for detecting AE typing as shown in SEQ ID NOs: 1, 2 and 3; Upstream primers, downstream primers and probes for detecting CE typing as shown in SEQ ID NOs: 1, 2 and 4; Universal detection upstream primers, downstream primers and probes as shown in SEQ ID NOs: 5, 6 and 7; and a second nucleic acid composition comprising a primer probe set as shown below: Upstream primers, downstream primers and probes for detecting emr-miR10-5p as shown in SEQ ID NOs: 8, 9 and 10; The upstream primers, downstream primers and probe for detecting universal EG-let7-5p are shown in SEQ ID NOs: 11, 12 and 13.

5. The composition according to claim 1, characterized in that The composition further comprises: The third nucleic acid composition includes a primer probe set as shown below: The upstream primer, downstream primer and probe for detecting the internal standard are shown in SEQ ID NOs: 14, 15 and 16.

6. The composition according to any one of claims 1 to 5, characterized in that The fluorescent reporter group of the probe for AE typing and emr-miR10-5p is FAM; the fluorescent reporter group of the probe for CE typing and EG-let7-5p is ROX, and the fluorescent reporter group of the universal probe is CY5.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of a kit for detecting and typing the pathogen of echinococcosis.

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

9. The kit according to claim 8, characterized in that The kit also includes: at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, and a nucleic acid amplifying reagent.

10. Use of a composition for preparing a reagent for detecting and typing the pathogen of echinococcosis, wherein the detection comprises 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 to 6 or the kit according to any one of claims 7 to 8; 3) Obtain and analyze the results.

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