Serum circulating miRNA-based echinococcosis detection markers and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly discloses a pair of echinococcosis detection markers based on serum circulating miRNA and application of the echinococcosis detection markers, the detection markers are a combination of miR-192-5p and miR-122-5p, the abundance of the miR-192-5p and the miR-122-5p in the serum of an echinococcosis patient is remarkably reduced, the miR-192-5p and the miR-122-5p are in negative correlation with echinococcosis infection, and the threshold values are 0.35 and 0.04 respectively. During detection, the relative expression level (taking caenorhabditis elegans miR-39-3p as an external reference) of caenorhabditis elegans miR-39-3p in serum is determined through qPCR, and the positive and negative properties are judged according to a threshold value; the area under the ROC curve of the marker combination reaches 0.97, and a double-blind test shows that the positive rate and the negative rate of human and mouse serum detection are 100%. The method solves the problems of missed diagnosis and misdiagnosis in existing echinococcosis detection, can be used for preparing a detection kit, realizes accurate detection, curative effect evaluation and prognosis management of echinococcosis, and has extremely high clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to a pair of detection markers for echinococcosis based on serum circulating miRNA and application thereof. BACKGROUND

[0002] Echinococcosis (also known as hydatidosis) is a zoonosis caused by the larval stage of the tapeworm Echinococcus granulosus (hydatid cyst) parasitizing the liver of humans and animals, and is distributed worldwide. In endemic areas, the annual incidence is 0.03 to 1.2 / 100,000, and the mortality rate of patients with untreated or untimely treated echinococcosis is more than 90% within 10 to 15 years after infection. The World Health Organization has listed hydatidosis as one of the 17 neglected diseases to be controlled or eliminated by 2050.

[0003] The incubation period of echinococcosis is 10 to 15 years, so most patients seek treatment only after the disease has progressed to an advanced stage. Patients usually do not show clinical symptoms until the cysts caused by hydatid cysts reach a diameter of more than 10 cm in the liver or the volume of an organ is occupied by one or more cysts by more than 70%. The huge cysts cause physical compression or damage to the bile duct, hepatic vein, portal vein, or hepatic artery, causing liver tissue to atrophy and posing a threat to life. The treatment options for echinococcosis are limited, mainly including surgical intervention and drug deworming. However, in most cases, the parasite invades the intrahepatic structures such as blood vessels and bile ducts extensively, making surgery exceptionally complex and reducing the efficacy of deworming drugs, often leading to poor prognosis. For patients treated only with deworming agents, due to the large side effects of the drugs, rational drug use is also an important issue. Therefore, accurate detection and assessment of drug efficacy are key steps in the treatment and prognosis management of echinococcosis. Clinically, the detection of echinococcosis relies largely on conventional imaging techniques, which have high requirements for equipment and operating personnel. However, for cysts with a diameter of less than 2 cm, the detection rate is very high. At the same time, echinococcosis is easily misdiagnosed with diseases such as liver cysts, liver hemangiomas, and liver cancer. As an auxiliary detection method, immunological detection uses antigens derived from hydatid cyst fluid, protoscoleces, and other worm components, which are usually highly sensitive but lack specificity and have a high false positive rate.

[0004] The existing detection techniques have significant defects, including the following:

[0005] Imaging detection: relies on professional equipment and operating personnel, has a high detection rate for cysts with a diameter of less than 2 cm, and is difficult to distinguish between echinococcosis and diseases such as liver cysts, liver hemangiomas, and liver cancer, leading to misdiagnosis;

[0006] Immunological detection: uses hydatid cyst antigens (derived from cyst fluid, protoscoleces, etc.), which are highly sensitive but lack specificity and have a high false positive rate.

[0007] MicroRNAs (miRNAs) are a class of endogenous non-coding RNA molecules with a length of about 18-24 nucleotides, which regulate gene expression by binding to target mRNA and play an important role in various physiological and pathological processes. In recent years, studies have found that the abundance of circulating miRNAs in serum is closely related to the pathological and infectious state of the animal body, and can be used as a biomarker for various diseases, with the advantages of convenient detection, high sensitivity and strong specificity. Based on the characteristics of serum circulating miRNAs, it is expected to screen serum circulating miRNAs that can be used as markers for detecting hydatid disease, so as to improve the sensitivity and specificity of detection. SUMMARY

[0008] In view of the deficiencies of the prior art, the purpose of the present application is to provide a pair of hydatid disease detection markers based on serum circulating miRNAs and applications thereof, in order to solve the problems of missed diagnosis and misdiagnosis in the detection of hydatid disease by imaging and serological methods.

[0009] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0010] The pair of hydatid disease detection markers based on serum circulating miRNAs is a combination of miR-192-5p and miR-122-5p.

[0011] Preferably, the miRNAs in the combination of hydatid disease detection markers miR-192-5p and miR-122-5p are all miRNAs in serum.

[0012] Preferably, the construction method of the hydatid disease detection marker in the hydatid disease detection comprises the following steps:

[0013] Step 1: Establish a qPCR reaction system, and then use a qPCR instrument to perform a reaction, and the reaction conditions are: 95℃ for 30s, followed by 95℃ for 10s and 60℃ for 30s, for a total of 40 cycles;

[0014] Step 2: Calculate the relative expression levels of miR-192-5p and miR-122-5p in the serum to be tested, and determine the results, wherein the determination results are specifically: the threshold values of miR-192-5p and miR-122-5p are 0.35 and 0.04 respectively, if the relative expression level of any miRNA in the detected serum miR-192-5p and miR-122-5p is less than the threshold value, the serum to be tested can be determined as hydatid disease positive serum; if the relative expression levels of miR-192-5p and miR-122-5p in the serum to be tested are equal to or greater than the threshold values, the serum to be tested can be determined as hydatid disease negative serum.

[0015] Preferably, the application of the pair of serum circulating miRNA-based echinococcosis detection markers comprises the application in an echinococcosis detection kit and a detection drug.

[0016] Preferably, the application method of the echinococcosis detection markers in the detection kit and the detection drug comprises the following steps:

[0017] Step 1: total RNA of a serum sample to be detected is extracted, and Caenorhabditis elegans miR-39-3p is added as an external reference before extraction;

[0018] Step 2: using the total RNA in step 1 as a template, cDNA is synthesized using a reverse transcription primer, and a qPCR reaction system is established using a miR-192-5p primer, a miR-122-5p primer and a universal reverse primer, and an amplification reaction is performed by a qPCR instrument; the qPCR reaction conditions are as follows: 95℃ pre-denaturation for 30s, followed by 40 cycles, each cycle including 95℃ denaturation for 10s and 60℃ annealing and extension for 30s;

[0019] Step 3: using the external reference in step 1 as a reference, the relative expression levels of miR-192-5p and miR-122-5p in the serum to be detected are calculated by a 2-ΔΔCt method, and the results are determined according to a threshold value: if the relative expression level of miR-192-5p in the serum to be detected is less than 0.35, or / and the relative expression level of miR-122-5p is less than 0.04, it is determined that the serum to be detected is echinococcosis positive serum; if the relative expression levels of both are greater than or equal to the corresponding threshold value, it is determined that the serum to be detected is echinococcosis negative serum.

[0020] The beneficial effects of the present application are as follows:

[0021] The present application takes the established animal model sample as a detection basis, screens out a biomarker combination composed of miR-192-5p and miR-122-5p from the miRNAs combined with Argonaute2 in the liver cells of echinococcosis mice, constructs a standard qPCR amplification curve to guarantee detection accuracy, and uses various statistical methods to evaluate detection performance, thereby establishing an effective echinococcosis detection method, providing a high specificity and high sensitivity means for echinococcosis detection, treatment monitoring and prognosis evaluation, solving the problems of missed diagnosis and misdiagnosis in the existing detection methods, and thereby improving the effectiveness of echinococcosis detection, which specifically includes the following contents:

[0022] 1. Excellent detection performance

[0023] The area under the ROC curve (AUC) of the biomarker combination reached 0.97. Double-blind trials showed that the positive and negative rates of human and mouse serum detection were both 100%, completely solving the problems of missed diagnosis and misdiagnosis.

[0024] 2. Convenient and efficient operation

[0025] Based on qPCR technology, no complex equipment is required, and the detection cycle is short (approximately 1.5 hours for a single sample qPCR reaction), making it suitable for batch clinical testing.

[0026] 3. Good repeatability and stability

[0027] The qPCR amplification efficiencies of miR-192-5p and miR-122-5p were 85.6% and 87.9%, respectively, with intra-assay precision <7%, inter-assay precision <19%, and coefficients of variation ranging from 0.06% to 1.5%.

[0028] 4. Wide range of applicable scenarios

[0029] It can be used not only for the initial screening of echinococcosis, but also to assess the efficacy of drugs by monitoring changes in biomarker expression levels (such as the rebound of biomarker expression after oral albendazole), providing a basis for prognostic management. Attached Figure Description

[0030] Figure 1 Expression levels of serum circulating miR-192-5p and miR-122-5p during Echinococcus multilocularis infection;

[0031] Figure 2 Expression levels of miR-192-5p and miR-122-5p in the serum of mice infected with albendazole after oral administration;

[0032] Figure 3 qPCR amplification efficiency targeting miR-192-5p and miR-122-5p;

[0033] Figure 4 Linear correlation analysis of the abundance of miR-192-5p and miR-122-5p in AE-positive serum after serial dilution of AE-negative serum;

[0034] Figure 5 ROC curve analysis of the combination of miR-192-5p and miR-122-5p. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments:

[0036] The core technical solution of this invention revolves around "detection markers, detection methods, and application products," as detailed below:

[0037] (1) Biomarkers for echinococcosis detection

[0038] The combination of miR-192-5p and miR-122-5p was selected as a biomarker for echinococcosis detection. This combination is derived from circulating serum miRNAs, and its abundance is significantly negatively correlated with Echinococcus granulosus infection: the expression levels of both are significantly downregulated in the infected state and rebound after drug treatment; by setting thresholds (miR-192-5p = 0.35 and miR-122-5p = 0.04), echinococcosis-positive and echinococcosis-negative sera can be accurately distinguished.

[0039] (2) Methods for detecting echinococcosis

[0040] The core steps of establishing a qPCR detection method based on the above biomarkers include:

[0041] Step 1: Sample Pretreatment

[0042] Take 200 μL of serum to be tested, add 5 μL of 25 fmol of Caenorhabditis elegans miR-39-3p as an external reference, and extract total RNA using the Trizol method (A260 / A280 should meet the requirement of 1.8-2.2).

[0043] Step 2, cDNA Synthesis

[0044] Take 2 μg of total RNA, add 1 μL of reverse transcription primer (SEQ ID NO.1), and make up to 12 μL with DEPC water. Incubate at 65℃ for 5 min, then place on ice. Add 4 μL of 5× reverse transcription buffer, 2 μL of 10 mM dNTP mixture, 1 μL of reverse transcriptase, and 1 μL of RNase inhibitor. React at 16℃ for 30 min, 42℃ for 30 min, and 85℃ for 5 min. Dilute the product 10 times and store at -20℃.

[0045] Step 3: qPCR amplification and result interpretation

[0046] Prepare the reaction solution according to the qPCR reaction system, using miR-39-3p as a reference, and employ 2... -ΔΔCt The relative expression levels of miR-192-5p and miR-122-5p in the serum to be tested were calculated, and the results were determined based on the threshold.

[0047] (3) Application of markers

[0048] The application of biomarkers for echinococcosis detection includes their use in diagnostic kits and drugs for echinococcosis detection.

[0049] Example 1: Screening of AGO2-binding miRNAs in hepatocytes

[0050] Experimental techniques and methods:

[0051] 1. Echinococcus multilocularis mouse infection model: 6-8 week old BALB / c mice were selected and intraperitoneally injected with a suspension of Echinococcus multilocularis protoscolex (about 2000 protoscolex / mouse) to establish an infection model. A saline control group was set up at the same time.

[0052] 2. Hepatocyte isolation: Mouse hepatocytes were isolated using the collagenase perfusion method and then purified by Percoll density gradient centrifugation.

[0053] 3. AGO2 Immunoprecipitation: RNA immunoprecipitation was performed using an AGO2-specific antibody to enrich miRNAs that bind to AGO2.

[0054] 4. High-throughput sequencing: RNA-seq was used to sequence and analyze the isolated and purified miRNA samples that interact with Argonaute 2.

[0055] Experimental results:

[0056] Each sample yielded an average of 33.6M data points, with an average genome alignment rate of 6.5%. A total of 398 miRNAs were detected, with 28 showing differential expression. Among them, miR-34c-3p, miR-470-5p, and miR-465a-3p were upregulated, while the others, including miR-192-5p and miR-122-5p, were downregulated, as shown in Table 1.

[0057] Table 1. Differentially expressed AGO2-binding miRNAs in hepatocytes

[0058]

[0059] Example 2 Abundance analysis of serum circulating miR-192-5p and miR-122-5p during Echinococcus granulosus infection

[0060] Experimental techniques and methods:

[0061] 1. miRNA screening: Potential echinococcosis miRNA markers were screened according to the following criteria: potential echinococcosis miRNA markers should have a sequence read >100 in the control or infected group, be specifically or highly expressed in the liver, and have a qPCR Ct value <34.

[0062] 2. Serum Total RNA Extraction: 200 μL of serum samples from mice infected with Echinococcus multilocularis at different stages were collected, and 5 μL of miDETECT™ miRNA (cel-miR-39-3p, 25 fmol) was added as an internal control. Total RNA was extracted using Trizol, and RNA concentration and purity were detected using NanoDrop. The A260 / A280 ratio of RNA should be between 1.8 and 2.2.

[0063] 3. cDNA synthesis and qPCR reaction: Take 2 μg of total RNA and add 1 μL of reverse transcription primer. Make up to 12 μL with DEPC water, incubate at 65°C for 5 minutes, then place on ice. Add the following reverse transcription reaction mixture: 5× reverse transcription buffer: 4 μL, dNTP mixture (10mM): 2 μL, reverse transcriptase: 1 μL, RNase inhibitor: 1 μL. Reaction conditions: 16°C for 30 min, 42°C for 30 min, 85°C for 5 min. Dilute the cDNA product 10-fold and store at -20°C.

[0064] The qPCR system and reaction were performed as follows:

[0065]

[0066] After mixing, perform the qPCR reaction under the following conditions:

[0067]

[0068] The primer sequences are shown below:

[0069]

[0070] 4. Calculate the relative expression levels of miRNAs: Using cel-miR-39-3p as an internal reference, the relative expression levels of each miRNA were calculated using 2-ΔΔCt.

[0071] 5. Three months after infection with Echinococcus multilocularis, mice were given albendazole orally once a day for 15 consecutive days, then sacrificed. Serum was separated, and the relative expression levels of each miRNA in the serum were analyzed using the method described above.

[0072] 6. Data Analysis: Calculate the mean ± standard deviation of each miRNA in different groups. Differences in means between two groups were compared using a Student's t-test; for three or more groups, one-way ANOVA was used for comparison. A p-value < 0.05 was considered statistically significant.

[0073] Experimental results:

[0074] Based on the number of miRNA sequences (>100), Ct values ​​(<34), and expression levels in hepatocytes in the control and infection groups, miR-192-5p and miR-122-5p were screened and further analyzed. Currently, these two miRNAs have been found to be significantly elevated in the serum of patients with liver cancer and hepatitis, but significantly decreased during Echinococcus euphorbia infection (P <0.0001). Figure 1 Conversely, after 15 days of antiparasitic drug treatment, the abundance of these two miRNAs in serum gradually increased (P < 0.05). Figure 2 The above results indicate that the abundance of circulating miR-192-5p and miR-122-5p in serum is closely related to the viability of the parasite.

[0075] Example 3: Establishment of a qPCR detection method for echinococcosis based on circulating miR-192-5p and miR-122-5p in serum

[0076] Experimental methods:

[0077] 1. qPCR amplification efficiency and detection limit analysis: DNA fragments containing miR-192-5p and miR-122-5p sequences were designed, and PCR amplification was performed using A9 LongHiFi PCR premix. The specific reaction system is shown in the table below:

[0078]

[0079] PCR conditions were: 98°C for 3 min; 98°C for 10 s, 55°C for 15 s, 72°C for 30 s, 35 cycles; 72°C for 2 min. The PCR product was purified by agarose gel electrophoresis, cloned into the pTOPO-Blunt vector, transformed into competent cells, and plasmid DNA was extracted and sequenced for verification. Plasmid concentration (ng / μL) was determined using NanoDrop, and the copy number (copies / μL) was calculated using the formula: (concentration × 6.02 × 10²³) / (plasmid length × 650 × 10⁹). A 1 × 10⁸ copy plasmid was serially diluted 10-fold and used as a template for PCR reactions under the following conditions: 95°C for 30 s; 95°C for 10 s, 60°C for 30 s, 40 cycles.

[0080] 2. ROC curve analysis and threshold determination: Serum samples from 25 mice diagnosed with echinococcosis and 25 healthy mice were collected as controls. The relative expression levels of miR-192-5p and miR-122-5p in the two groups were determined using the qPCR method described above. ROC curves and Youden's index were analyzed using SPSS software.

[0081] Experimental results:

[0082] The qPCR amplification efficiencies of miR-192-5p and miR-122-5p were both acceptable, at 85.6% and 87.9%, respectively. Figure 3 Serial dilutions of AE-positive serum with AE-negative serum further demonstrated that each qPCR reaction exhibited effective amplification and a significant linear correlation (R² > 0.94). Figure 4 The detection limit of miR-192-5p-based qPCR was 100 copies / µL, while that of miR-122-5p-based qPCR was 10 copies / µL; both had low coefficients of variation (CV), ranging from 0.06% to 1.5%. Their intra-assay precision was less than 7%, while the inter-assay precision of miR-122-5p was 8.7%–12.8%, and that of miR-192-5p was 13.1%–18.3%. These results indicate that qPCR targeting miR-192-5p and miR-122-5p exhibits good performance in terms of amplification and reproducibility.

[0083] ROC curve analysis revealed that the area under the curve (AUC) of the qPCR method based on the combination of miR-192-5p and miR-122-5p was ≥ 0.97. Figure 5 This indicates that the method has excellent detection performance. When the Youden index is at its maximum, the relative expression levels of miR-192-5p and miR-122-5p are 0.35 and 0.04, respectively. Therefore, their thresholds are set to 0.35 and 0.04, respectively. That is, when the relative expression level of miR-192-5p in the serum to be tested is less than 0.35 and / or the relative expression level of miR-122-5p is less than 0.04, the serum to be tested can be identified as positive for echinococcosis; if the relative expression levels of miR-192-5p and miR-122-5p in the serum to be tested are not less than their thresholds, the serum to be tested can be identified as negative for echinococcosis.

[0084] Example 4: Clinical Sample Validation

[0085] Experimental methods:

[0086] 1. Clinical samples: A total of 117 human serum samples were collected, including 58 serum samples from patients with echinococcosis and 59 serum samples from healthy controls; a total of 50 mouse serum samples were collected, including 25 serum samples from mice infected with Echinococcus multilocularis and 25 serum samples from healthy controls.

[0087] 2. Double-blind validation: All samples were randomly coded by third-party researchers, and the testing personnel were unaware of the actual grouping information of the samples. Using the qPCR method established in Example 3, the testing personnel analyzed all samples and calculated the relative expression levels of miR-192-5p and miR-122-5p in the serum to be tested. After completion, the results were unblinded and analyzed uniformly.

[0088] Experimental results:

[0089] The results showed that, for double-blind human serum, the detection rates of echinococcosis-positive serum (58 / 58) and echinococcosis-negative serum (59 / 59) were both 100% using the qPCR method based on the combination of miR-192-5p and miR-122-5p; for double-blind mouse serum, the detection rates of echinococcosis-positive serum (25 / 25) and echinococcosis-negative serum (25 / 25) were also both 100%.

[0090] The results of the above examples indicate that the combination of serum circulating miR-192-5p and miR-122-5p is an effective biomarker for echinococcosis.

[0091] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pair of biomarkers for echinococcosis detection based on circulating miRNAs in serum, characterized in that, The biomarkers for echinococcosis detection are a combination of miR-192-5p and miR-122-5p.

2. The pair of echinococcosis detection biomarkers based on serum circulating miRNAs according to claim 1, characterized in that, The miRNAs in the miR-192-5p and miR-122-5p biomarker combinations for echinococcosis detection are all serum miRNAs.

3. The pair of echinococcosis detection biomarkers based on serum circulating miRNAs according to claim 1 or 2, characterized in that: The method for constructing the echinococcosis detection biomarker in echinococcosis detection includes the following steps: Step 1: Establish a qPCR reaction system, and then use a qPCR instrument to carry out the reaction. The reaction conditions are: 95℃ for 30 s, then 95℃ for 10 s and 60℃ for 30 s, for a total of 40 cycles. Step 2: Calculate the relative expression levels of miR-192-5p and miR-122-5p in the serum to be tested, and determine the results. Specifically, the thresholds for miR-192-5p and miR-122-5p are 0.35 and 0.04, respectively. If the relative expression level of either miRNA in the serum is less than its threshold, the serum to be tested can be determined to be positive for echinococcosis. If the relative expression levels of miR-192-5p and miR-122-5p in the serum to be tested are equal to or greater than their thresholds, the serum to be tested can be determined to be negative for echinococcosis.

4. The application of the pair of serum circulating miRNA-based biomarkers for echinococcosis detection according to claim 3, characterized in that: The application of the biomarkers for echinococcosis detection includes their use in detection kits and drugs for echinococcosis detection.

5. The application of the pair of serum circulating miRNA-based biomarkers for echinococcosis detection according to claim 4, characterized in that... The method for applying the echinococcosis detection biomarker in the detection kit and detection drug includes the following steps: Step 1: Extract total RNA from the serum sample to be tested. Before extraction, add Caenorhabditis elegans miR-39-3p to the serum sample as an external reference. Step 2: Using the total RNA described in Step 1 as a template, cDNA was synthesized using reverse transcription primers. Then, a qPCR reaction system was established using miR-192-5p primers, miR-122-5p primers, and universal reverse primers. The amplification reaction was performed using a qPCR instrument. The qPCR reaction conditions were: 95℃ pre-denaturation for 30s, followed by 40 cycles, each cycle including 95℃ denaturation for 10s and 60℃ annealing extension for 30s. Step 3: Using the external parameters described in Step 1 as a reference, adopt 2 -ΔΔCt The relative expression levels of miR-192-5p and miR-122-5p in the serum to be tested were calculated, and the results were judged according to the threshold: if the relative expression level of miR-192-5p in the serum to be tested was less than 0.35, or / and the relative expression level of miR-122-5p was less than 0.04, it was judged as echinococcosis positive serum; if the relative expression levels of both were greater than or equal to the corresponding threshold, it was judged as echinococcosis negative serum.

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