Kit for early differential diagnosis of EBV-HLH and method for auxiliary diagnosis of EBV-HLH

Abstract

The invention relates to a kit for early differential diagnosis of EBV-HLH and a method for auxiliary diagnosis of EBV-HLH. The kit comprises an EBV nucleic acid detection reagent and a miRNA-375 detection reagent. The kit provided by the invention is used for combined detection of EBV-DNA and miRNA-375 in a detection sample, and when an EBV-DNA detection result is greater than 5000 copies / mL and a miRNA-375 detection CT value is less than 35, suspected EBV-HLH is diagnosed. The invention not only provides a specific diagnostic index for EB infected diseases, but also serves as a reliable auxiliary diagnostic tool for EBV-HLH and other EB virus infection related diseases. When the kit provided by the invention is used for identifying and diagnosing EBV-HLH, the sensitivity and the accuracy are higher.

Description

Technical Field

[0001] This disclosure relates to the field of biotechnology, specifically to a kit for the early differential diagnosis of EBV-HLH and a method for assisting in the diagnosis of EBV-HLH. Background Technology

[0002] Epstein-Barr virus (EBV) is a DNA virus widely present in the human population, generally in a latent infection state, which can become active when the host's immune function is weakened. EBV is an important tumor-associated virus, closely related not only to the development of certain types of neoplastic diseases, such as nasopharyngeal carcinoma, gastric cancer, and lymphoma, but also to non-neoplastic diseases, including infectious mononucleosis (IM) caused by primary EBV infection, chronic active Epstein-Barr virus infection (CAEBV), and Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH). This viral infection is particularly common in children and adolescents, with most individuals acquiring lifelong immunity after initial infection. However, for individuals with weakened immune systems, EBV infection can cause more serious health problems, especially posing a serious threat to the life and health of children.

[0003] Both intraepithelial neoplasia (IM) and EBV-HLH are caused by EBV infection, but they are two distinct disease states with different clinical presentations and treatment approaches. IM is usually a benign, self-limiting disease. Due to its relatively mild symptoms, treatment is primarily symptomatic and supportive, and usually does not require specific therapies. Antiviral therapy may be used when necessary, and symptoms often resolve spontaneously within a few weeks, with a generally good prognosis. In contrast, EBV-HLH triggers an abnormal immune response, leading to severe systemic symptoms and multiple organ dysfunction, classifying it as a life-threatening disease requiring urgent intervention. Therefore, in addition to routine symptomatic and supportive care, treatment for EBV-HLH mainly relies on immunosuppressive therapy, such as hormones or chemotherapy. Some children require emergency hematopoietic stem cell transplantation (HSCT). However, EBV-HLH progresses rapidly, and if a cytokine storm with multiple organ failure occurs in the late stages, the condition is often irreversible. Without timely and effective treatment, the survival time for severe cases may be less than two months. Therefore, early and accurate differential diagnosis between EBV-HLH and IM is of great significance for selecting appropriate treatment plans and predicting prognosis.

[0004] In existing technologies, the differential diagnosis between EBV HLH and EBV IM mainly employs a combination of clinical symptoms and multiple clinical biochemical indicators, such as:

[0005] An existing technical solution uses a predictive model based on clinical symptoms and laboratory test results of four biomarkers (IL-10, IFN-γ, ferritin and D-dimer levels) to distinguish between EBV-HLH and EBV-IM.

[0006] When using the criteria of interleukin (IL)-10 > 89.6 pg / mL, interferon (IFN)-γ > 45.6 pg / mL, ferritin > 429 μg / L, D-dimer > 3.15 mg / L, and triglycerides > 2.1 mmol / L, the sensitivities for predicting EBV-HLH were 87.9%, 90.7%, 98.1%, 91.1%, and 81.5%, respectively, and the specificities were 98.4%, 96.4%, 91.1%, and 81.5%, respectively. Based on this model, the sensitivities for predicting EBV-HLH were 87.9%, 90.7%, 98.1%, 91.1%, and 81.5%, respectively, and the specificities were 98.4%, 96.3%, 96.5%, 94.1%, and 80.6%, respectively. A logistic regression model based on four parameters (IL-10, ferritin, D-dimer, and triglycerides) was established to distinguish between EBV-HLH patients and EBV-IM patients, with a sensitivity of 98.0% and a specificity of 98.2%.

[0007] (Reference: Cai L, Xing Y, Xia Y, Zhang Z, Luo Z, Tang Y, Chen Y, Xu 26;23(1):728.doi:10.1186 / s12879-023-08654-6.PMID:37880605; PMCID:PMC10601177)

[0008] Another technical approach uses total bilirubin and triglycerides as indicators to effectively assess the efficacy and prognosis of children in the EVB HLH induction period.

[0009] Ninety-five children with hemophagocytic lymphocytosis (HLH) were included in this study, of whom 43 (45.3%) had EBV-HLH and 52 (54.7%) did not. Laboratory results showed that the EBV-HLH group had a significantly lower absolute neutrophil count (ANC) (P = 0.031) and higher triglyceride (TG) levels (P = 0.036) compared to the non-EBV-HLH group. The remission rates during the induction phase were 75.8% in the EBV-HLH group and 89.3% in the non-EBV-HLH group. Correlation analysis showed that elevated levels of total bilirubin (TBIL) (P = 0.042), triglycerides (TG) (P = 0.009), serum ferritin (SF) (P = 0.008), and interleukin-8 (IL-8) (P = 0.004) were associated with a higher remission rate during the induction phase in the EBV-HLH group. Further univariate analysis showed that elevated TBIL (P = 0.048) and TG (P = 0.019) were significant risk factors for remission rate during induction in the EBV-HLH group. In multivariate analysis, a statistically significant difference in TG elevation was observed between the two groups (P = 0.015). Correlation analysis showed that elevated TBIL (P = 0.030), elevated SF (P = 0.020), and elevated interleukin-6 (P = 0.010) were associated with induction mortality in children with EBV-HLH.

[0010] (Reference: Qiu KY, Guo SY, Zeng YH, Liao XY, Lin SF, Fang JP, ZhouDH. Analysis of clinical characteristics and prognostic factors associated with EBV-associated HLH in children.Hematology.2022Dec;27(1):874-880.doi:10.1080 / 16078454.2022.2109328.PMID:35950974)

[0011] It is evident that current technologies for detecting multiple diagnostic indicators are time-consuming and lack disease specificity. Clinically, a definitive diagnosis of EBV-HLH requires simultaneous fulfillment of both the diagnostic criteria for HLH and indicators of EBV infection. Suspected HLH cases should have all HLH confirmatory and etiological examinations completed as quickly as possible (24–48 hours), along with monitoring of HLH-related indicators. Treatment should only begin immediately if the diagnostic criteria are met, or if the criteria are not fully met but the case is highly suspected and the condition is rapidly deteriorating (with some HLH-related test results not yet available). Therefore, current technologies cannot provide timely and effective diagnosis and treatment for EBV-HLH.

[0012] In conclusion, early differential diagnosis between IM and HLH is crucial for improving the diagnostic accuracy and treatment success rate of HLH. Currently, there is a lack of differential diagnostic indicators that can accurately differentiate between EBV IM and EBV HLH at an early stage. Summary of the Invention

[0013] In view of this, the purpose of this disclosure is to solve at least one of the problems existing in the prior art, and more specifically, this disclosure aims to provide a differential diagnostic index for the early differential diagnosis of EBV HLH.

[0014] This disclosure proposes a detection biomarker for the early differential diagnosis of EBV-HLH, comprising EBV-DNA and miRNA-375. Among them, miRNA-375, as a novel differential diagnostic indicator, has high specificity in the diagnosis of EBV-HLH and can more accurately determine the stage of EBV infection, providing strong support for clinical diagnosis and treatment.

[0015] Furthermore, in a first aspect of the present disclosure, a kit for early differential diagnosis of EBV-HLH is provided, comprising an EBV nucleic acid detection reagent and a miRNA-375 detection reagent.

[0016] By using EBV nucleic acid detection reagents and miRNA-375 detection reagents, not only can the EBV-DNA content in the test samples be quantitatively analyzed, but more importantly, the expression level of miRNA-375 in the test samples can be analyzed. These data provide crucial intermediate evidence for the identification and diagnosis of EBV-related infections. Specifically, accurate quantification of EBV-DNA provides direct information on viral load; while the specific expression pattern of miRNA-375 serves as a biomarker, providing strong diagnostic support for accurately distinguishing EBV-HLH. The combined detection of EBV-DNA and miRNA-375 in the kit enhances the sensitivity and accuracy of EBV infection diagnosis.

[0017] Optionally, the miRNA-375 detection reagent includes primers that can specifically bind to miRNA-375.

[0018] Preferably, the primers include primers for hsa-miR-375.

[0019] The primer sequence is: 5'-TTTGTTCGTTCGGCTCGCG-3'.

[0020] Primers were designed based on the sequence characteristics of miRNA-375 to ensure high specificity and binding ability, achieving precise amplification of miRNA-375 and further enhancing the sensitivity and specificity of EBV-HLH diagnosis.

[0021] Optionally, the miRNA-375 detection kit also includes plasma total RNA extraction reagent, miRNA reverse transcription reagent, and qPCR reaction reagent.

[0022] Optionally, the EBV nucleic acid detection reagent includes EBV nucleic acid extraction reagent, PCR reaction solution, and EBV quality control solution.

[0023] Preferably, the EBV control solution includes a negative control, a critical positive control solution, and a positive quantitative reference solution. More preferably, the content of the positive quantitative reference solution is 10. 2 -10 7 Copy / ml.

[0024] Optionally, the plasma total RNA extraction reagents include denaturation buffer, cel-miR-39 external reference, acid-phenol-chloroform solution, anhydrous ethanol, and elution buffer.

[0025] Optionally, the miRNA reverse transcription reagent includes a reverse transcription mixture and a reverse transcriptase.

[0026] Optionally, the qPCR reaction reagent includes an amplification mixture.

[0027] The real-time quantitative PCR (qPCR) method was used to quantitatively detect miRNA-375, obtaining further reliable data on the expression level of miRNA-375 and improving the accuracy of detection.

[0028] Optionally, the kit is applicable to test samples including plasma separated using sodium citrate anticoagulant tubes. This disclosed kit has broad applicability and can be used for a wide variety of test samples.

[0029] Secondly, this disclosure provides a method for assisting in the diagnosis of EBV-HLH, in which all steps are performed by a computer or other device, including:

[0030] Obtain the EBV nucleic acid test results of the sample;

[0031] Obtain the CT value of miRNA-375 in the sample;

[0032] When the EBV-DNA test result is greater than 5000 copies / mL and the miRNA-375 test CT value is less than 35, auxiliary diagnostic result information of suspected EBV-HLH is output.

[0033] The aforementioned approach proposes an auxiliary diagnostic method that analyzes the detection results of EB-DNA and plasma miR-375 using computational equipment, aiming to improve the accuracy of EBV-HLH diagnosis. Using this method, the area under the characteristic curve (ROC) (AUC) can reach 0.951 or higher, demonstrating the excellent diagnostic performance of this auxiliary diagnostic method.

[0034] In the detection system that includes the aforementioned kits and auxiliary diagnostic methods, using miRNA-375 as a key biomarker not only provides a more accurate diagnostic tool, but also significantly improves the diagnostic accuracy and specificity of EBV-HLH compared to single or multiple detection indicators. This optimizes the diagnostic process for EBV-HLH, promotes the further development of clinical diagnostic technology for EBV-HLH, provides doctors with more accurate diagnostic evidence, helps to develop more personalized treatment plans, and ultimately improves patients' quality of life and prognosis. Attached Figure Description

[0035] The above and other features, advantages, and aspects of the embodiments of this disclosure will become more apparent from the accompanying drawings and the following detailed description. In the drawings, the same or similar reference numerals denote the same or similar elements, wherein:

[0036] Figure 1 A schematic diagram showing the enrichment of target genes of three miRNAs in the miRNAs in cancer pathway.

[0037] Figure 2 A schematic diagram showing the differential expression of three specific proteins in the IM group and the EBV-HLH group;

[0038] Figure 3 A diagram showing the diagnostic efficacy of different diagnostic indicators, either individually or in combination. Detailed Implementation

[0039] To date, there are no differential diagnostic indicators for early differential diagnosis of EBV HLH. This disclosure intends to publish a differential diagnostic indicator for EBV HLH.

[0040] Plasma proteomics analysis was performed using liquid chromatography-mass spectrometry (LC-MS) to identify specific proteins for EBV-IM and EBV-HLH. Pathway analysis was also conducted on upregulated proteins in patients with EBV-IM and EBV-HLH. Compared to healthy controls, 63 and 18 proteins, respectively, were upregulated in patients with EBV-IM and EBV-HLH. Pathway and process enrichment analysis showed that the complement system was the most enriched upregulated protein category in EBV-IM, while proteins related to immune effector processes were most enriched in EBV-HLH. Of the 18 upregulated proteins in EBV-HLH, 7 were unique to EBV-HLH. These specific proteins are associated with three pathways, while lipoprotein E was found in all pathways. Proteomics analysis can provide new perspectives for understanding the host response to EBV infection and the pathogenesis of EBV-related diseases.

[0041] like Figure 1-2 As shown, based on the host's response to EBV infection and the pathogenesis of EBV-related diseases, the inventors, through proteomics analysis, discovered that the target genes of three miRNAs are enriched in the viral oncogenic pathway. Furthermore, the inventors pioneered the discovery that miR-375 and miR-148a-3p expression differed between the IM group and the EBV-HLH group, therefore choosing miR-375 and miR-148a-3p as detection biomarkers. Figure 2 Based on this, the inventors discovered that compared to the diagnosis of biomarkers alone, the combined detection of miR-375 and EBV DNA has higher specificity, sensitivity, and accuracy. Therefore, miR-375 and EBV DNA were chosen as the detection indicators.

[0042] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. The embodiments and descriptions of this invention are only for explaining this invention, but these embodiments and descriptions do not limit the scope of this invention.

[0043] It should be understood that the order of steps or the sequence of actions is not important as long as this disclosure remains operational. Furthermore, two or more steps or actions may be performed simultaneously.

[0044] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate this disclosure and does not constitute a limitation on the scope of this disclosure unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this disclosure.

[0045] Unless otherwise specified, all terms used herein, including technical and scientific terms, shall have the same meaning as commonly understood by one of ordinary skill in the art. Terms defined in commonly used dictionaries shall be interpreted as having the same meaning as in the relevant technical context, and shall not be construed as having a formal meaning in an idealized or overly formal sense unless expressly defined in the specification.

[0046] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0047] All reagents, raw materials, instruments or equipment mentioned below can be purchased and obtained from the market.

[0048] In one embodiment, a kit for early differential diagnosis of EBV-HLH is prepared, comprising an EBV nucleic acid detection reagent and a miRNA-375 detection reagent, for detecting the EBV-DNA content and miRNA-375 expression level in the treated test sample.

[0049] In one specific embodiment, the miRNA-375 detection reagents in the kit include plasma total RNA extraction reagent, miRNA reverse transcription reagent, and qPCR reaction reagent, which are used to extract total RNA from plasma, obtain cDNA, and quantitatively detect miRNA-375, respectively. It should be understood that those skilled in the art can also use other reagents capable of detecting the expression level of miRNA-375 in the sample.

[0050] Optionally, the miRNA-375 detection reagent may also include primers that can specifically bind to miRNA-375.

[0051] In one specific embodiment, the primers used in the kit are hsa-miR-375 primers, with the primer sequence being: 5'-TTTGTTCGTTCGGCTCGCG-3'.

[0052] In one specific embodiment, the kit may not contain primers, which may be designed and synthesized by the user or purchased separately.

[0053] In one specific embodiment, the plasma total RNA extraction reagent in the kit consists of denaturing buffer, cel-miR-39 external reference, acid-phenol-chloroform solution, anhydrous ethanol, and elution buffer.

[0054] In one specific embodiment, the miRNA reverse transcription reagent in the kit is selected from reverse transcription mixture and reverse transcriptase.

[0055] In one specific embodiment, the qPCR reaction reagents in the kit consist of an amplification mixture and redistilled water.

[0056] In one specific embodiment, the EBV nucleic acid detection reagent in the kit includes EBV nucleic acid extraction reagent, PCR reaction solution, and EBV quality control solution. More preferably, the EBV quality control solution includes negative quality control, borderline positive quality control solution, and positive quantitative reference solution, which are used to detect the EBV-DNA content in the sample.

[0057] In another specific embodiment, the EBV nucleic acid detection reagent in the kit is an EBV nucleic acid amplification fluorescent detection reagent. It should be understood that those skilled in the art can also use other reagents capable of quantitatively detecting EBV-DNA in the sample.

[0058] The following steps were taken to detect EBV-DNA and miRNA-375 in the sample using the above kit:

[0059] 1. Before detection, the sample is processed and the instrument is set. In this embodiment, an ABI 7300 real-time quantitative PCR amplification instrument is used, and the performance parameters are set as follows: detection limit 5.0 × 10⁻⁶. 2 Copies / ml, linear range 5.0 × 10⁻⁶ 2 ~5.0×10 8 Copy / ml.

[0060] Before using the kit disclosed herein to test samples, the samples must be collected and preserved, and the samples should meet the testing standards. In a specific embodiment, this kit is applicable to plasma test samples separated using sodium citrate anticoagulant tubes, but this disclosure is not limited to this. In the embodiments, the kit is also applicable to other types of test samples such as whole blood, peripheral blood, serum, plasma, or nasopharyngeal swab suspensions.

[0061] In this embodiment, plasma separated using sodium citrate anticoagulant tubes was used as the test sample for the kit, and the sample was collected and preserved according to the following method. It should be understood that those skilled in the art can select appropriate processing methods based on the differences in the test samples; other sample collection and processing methods will not be described in detail here.

[0062] (1) Specimen collection: Clinical medical staff use a disposable vacuum blood collection device to draw 2-5 ml of venous blood from the subject and inject it into a disposable sodium citrate anticoagulant tube, which is then sealed and sent for testing. Centrifuge at 4000 rpm for 5 minutes using a regular centrifuge to separate the plasma for later use.

[0063] (2) Specimen preservation and transportation: Once collected, specimens should be sent for testing as soon as possible; specimens that can be used for testing immediately should not be stored at room temperature for more than 12 hours. Specimens that cannot be tested immediately should be stored at 2-8℃ for no more than 7 days, or stored at -20℃ for testing, with a shelf life of 6 months. Specimens should be transported over long distances using a 0℃ ice bath.

[0064] (3) For unqualified specimens (such as severely hemolyzed, lipemic specimens, heparin anticoagulated specimens, etc.), the clinic should be notified by telephone in a timely manner and the "Unqualified Specimen Rejection Registration Form" should be filled out. If the unqualified specimens are accepted at the request of the clinic, it should be noted in the remarks of the test report and a record should be made.

[0065] (4) Laboratory storage: Temperature: should be controlled within the range of 18 to 30℃; Humidity: should be controlled within the range of 20 to 80% RH; and each area should be operated independently and unidirectionally in strict accordance with the "Management Measures for Clinical Gene Amplification Laboratories" and "Work Specifications for Clinical Gene Amplification Laboratories" issued by the Ministry of Health of the People's Republic of China.

[0066] 2. Based on the highly conserved regions in the whole genome sequence of EBV, a pair of EBV-specific primers were designed, and EBV-DNA was quantitatively detected by PCR in vitro amplification, thereby enabling rapid early diagnosis of EBV infection.

[0067] In a specific implementation, the EBV-DNA content of qualified plasma samples is detected using the kit provided above. The specific detection procedure is as follows:

[0068] In this embodiment, the EBV nucleic acid detection reagent uses primers and a PCR reaction solution containing thermostable DNA polymerase (Taq polymerase) and nucleotide monomers (dNTPs). Optionally, the EBV primers are: upstream primer: 5'-AGGACCTAGCTCGCTGCCCTA-3', downstream primer: 5'-AAAACATGCGGACCACCA-3'; in another embodiment, an EBV nucleic acid amplification fluorescent detection reagent can also be used.

[0069] (1) Plasma sample processing: Take 50 μl of plasma and add it to a 0.5 ml disposable autoclaved centrifuge tube, and add an equal amount of DNA concentrate (purchased from Guangzhou Da An Gene Co., Ltd.). Incubate at 100℃ for 10 min; centrifuge at 12,000 rpm for 10 min, and set aside.

[0070] (2) Quality control solution processing:

[0071] Take out the EBV negative control sample, centrifuge at 8,000 rpm for a few seconds, aspirate 100 μl into a 0.5 ml disposable autoclaved centrifuge tube, add 100 μl of DNA extraction buffer, and vortex to mix; centrifuge at 12,000 rpm for 10 min; discard the supernatant, add 20 μl of DNA extraction buffer to the precipitate, vortex to mix, and incubate at 100℃ for 10 ± 1 min; centrifuge at 12,000 rpm for 5–10 min, and set aside.

[0072] Remove the EBV borderline positive control and process it as described above for the EBV negative control.

[0073] Take out the EBV positive quantitative reference sample: vortex to mix, centrifuge at 8,000 rpm for a few seconds, and set aside.

[0074] (3) PCR amplification:

[0075] Take several PCR reaction tubes and add 5 μl of supernatant from the processed samples (including plasma samples, EBV negative controls, and EBV borderline positive controls) and 2 μl of EBV positive quantitative reference material to each. Centrifuge briefly for a few seconds and place them in the instrument's sample chamber. Set the EBV negative controls, EBV positive quantitative reference material, and unknown samples (including borderline positive controls) in the corresponding order. The instrument's cycling conditions are as follows: 93℃ 2 min → 93℃ 45 s → 55℃ 60 s, 10 cycles; 93℃ 30 s → 55℃ 45 s, 30 cycles. Save the file and run the test.

[0076] The above amplification results were calculated and reported, and the EBV-DNA content was recorded.

[0077] EBV-DNA testing plays a crucial role in the treatment of intraepithelial neoplasia (IM) and EBV-HLH, and can be used as an adjunct in the differential diagnosis of other pathogens. For children with IM, viral load testing is performed, even if the disease course is within 2-3 weeks and there are recurrent episodes, before reaching 3 months, to diagnose CAEBV and EBV-HLH. This can prevent the disease from progressing to severe illness and help determine prognosis. For children with CAEBV and EBV-HLH, EBV DNA testing can also be used to monitor disease progression and evaluate treatment effectiveness, guiding clinical medication and preventing adverse reactions due to overdose.

[0078] To further improve the accuracy, specificity, and sensitivity of the detection, the expression level of miRNA-375 in qualified plasma samples was detected using the miRNA-375 detection reagent in the above kit.

[0079] 3. Design miRNA-375-specific primers and use qPCR to quantitatively detect miRNA-375 in plasma. The specific detection method is as follows:

[0080] (1) Extraction of total RNA from plasma:

[0081] Take 200 μL of the separated plasma and add it to an EP tube. Add 200 μL of 2X denaturing buffer (preheated at 37°C for at least 2 hours to fully dissolve it) and mix thoroughly. Let it stand on ice for 5 minutes, then add 3.75 μL of cel-miR-39 external reference (in this example, exogenous cel-miR-39 is used as a reference, purchased from Qiagen). Then add 400 μL of acid-phenol-chloroform solution and vortex for 60 seconds. After mixing thoroughly, centrifuge at 12000g for 5 minutes at room temperature. Then transfer the upper aqueous phase to a new collection tube, add 1.25 times the volume of anhydrous ethanol, and mix by inverting.

[0082] Transfer 700 μL of sample to the filter column, centrifuge at 10000 x g for 15 seconds at room temperature, and discard the liquid. Add 700 μL of miRNA elution buffer 1 to the filter column, centrifuge at 10000 x g for 15 seconds at room temperature, and discard the liquid. Add 2 / 3 of 500 μL of miRNA elution buffer to the filter column, centrifuge at 10000 x g for 15 seconds at room temperature, and discard the liquid. Add another 2 / 3 of 500 μL of miRNA elution buffer to the filter column, centrifuge at 10000 x g for 15 seconds at room temperature, and discard the liquid. Centrifuge at 10000 x g for 1 minute to remove any residual liquid.

[0083] Transfer the filter column to a new 1.5 mL collection tube, add 30 μL of elution buffer preheated to 95 °C to the center of the filter column, centrifuge at 10000 x g for 30 seconds, and collect the total RNA.

[0084] (2) miRNA reverse transcription:

[0085] The reaction program was set as follows: 37℃ for 60 minutes; 85℃ for 5 minutes; 4℃ indefinite cycle. The reaction system is shown in Table 1.

[0086] Table 1

[0087]

[0088]

[0089] (3) qPCR amplification:

[0090] In this embodiment, the miRNA primers were purchased from Guangzhou Ribo Biotechnology Co., Ltd., and cel-miR-39 was used as an external reference. The cDNA obtained by reverse transcription of the above miRNA was diluted in 90 μL of redistilled water, and the diluted cDNA was used as the template for qPCR reaction. The qPCR reaction system was prepared according to the proportions shown in Table 2. It should be understood that in other embodiments, the qPCR reaction system can also be adjusted according to requirements.

[0091] Table 2

[0092]

[0093] The reaction program is set as follows:

[0094] Step 1: 95℃ for 30 seconds;

[0095] Step Two:

[0096] GOTO: (6 loops)

[0097] 94℃ for 10 seconds

[0098] 45℃ for 30 seconds

[0099] 72℃ for 60 seconds

[0100] GOTO: (40 loops)

[0101] 95℃ for 5 seconds

[0102] 60℃ for 30 seconds

[0103] GOTO: (1 loop)

[0104] 95℃ for 5 seconds;

[0105] Step 3: Melt Curve.

[0106] Record the above miRNA-375 detection results and perform statistical analysis on the above amplification results.

[0107] 4. Interpretation of EBV-DNA and miRNA-375 detection results.

[0108] Analyze the EBV-DNA detection results: Adjust the start, stop, and threshold values ​​of the baseline based on the analyzed image (adjust as needed; the start value can be 1–10, and the stop value can be 5–20) to achieve the optimal standard curve in the Std curve window, i.e., a correlation value between -1.0 and -0.97. In the Results module, select Analyze to automatically analyze the results (C).

[0109] The test results of the quality control samples must simultaneously meet the following conditions:

[0110] Negative control: The growth curve does not show an S-shaped curve or the Ct value is 30.

[0111] Positive control: The growth curve shows an S-shaped curve, and the quantitative reference value for the critical positive control is within 2×10⁻⁶.2 Copies / ml ~ 2.0 × 10 4 Copy / ml range.

[0112] Positive quantitative reference material: The growth curve is S-shaped, Ct value < 27, and the linear correlation coefficient is 0.97 ≤ r ≤ 1.

[0113] The results of EBV-DNA testing are determined according to the following criteria:

[0114] (1) If the growth curve does not show an S-shaped curve or the Ct value is 30, then the total EB DNA content of the sample is judged to be less than the detection limit.

[0115] (2) If the sample's C < 5.00E+002, then the total EBV DNA content of the sample is below the detection limit.

[0116] (3) If 5.00E+002≤C≤5.00E+008 of the sample, then the total EBV DNA content of the sample = C copies / ml;

[0117] (4) If the sample's C > 5.00E+008, then the total EBV DNA content of the sample is > 5.0 × 10⁻⁶. 8 Copy / ml. If precise quantification is required, the extracted sample can be diluted to the linear range before detection.

[0118] Calculation method and reporting method: Based on the standard curve and Ct value, the EBV DNA test result is considered positive if it is greater than 5000 copies / mL, and the miRNA-375 test CT value is less than 35. Both are considered positive. The test kit is used to diagnose EBV HLH in plasma samples.

[0119] In this embodiment, the present disclosure provides a method for assisting in the diagnosis of EBV-HLH, wherein all steps of the method are implemented by a computer or other device, including:

[0120] Obtain the EBV nucleic acid test results of the sample;

[0121] Obtain the CT value of miRNA-375 in the sample;

[0122] Data processing was performed on the aforementioned nucleic acid test results and CT values. When the EBV DNA test result was greater than 5000 copies / mL and the miRNA-375 CT value was less than 35, auxiliary diagnostic results for suspected EBV-HLH were output.

[0123] In a typical embodiment, the aforementioned method for assisting in the diagnosis of EBV-HLH is executed by a computer device that includes at least an input module, a data processing module, and an output module.

[0124] The input module may include laboratory instruments capable of testing samples and acquiring EBV nucleic acid test results and miRNA-375 CT values, or data acquisition devices connected to storage devices storing the aforementioned test results of individuals seeking diagnosis. It may also be a manual input device such as a keyboard. The input module can transmit the acquired EBV nucleic acid test results and miRNA-375 CT values ​​to the data processing module via, for example, a bus.

[0125] The data processing module includes at least a processor and a storage device connected to the processor via, for example, a bus, in which a computer program is stored. When the processor is running, it can execute the aforementioned computer program to analyze the EBV nucleic acid detection results and miRNA-375 detection CT values ​​from the input module, and output auxiliary diagnostic result information to the output module based on the analysis results.

[0126] Typically, a computer program can output auxiliary diagnostic results for suspected EBV-HLH to the output module when the EBV nucleic acid test result is greater than 5000 copies / mL and the miRNA-375 CT value is less than 35.

[0127] The aforementioned output module may include a display device or similar device installed on the terminal to present the received auxiliary diagnostic results to the user. For example, it may present the auxiliary diagnostic results of suspected EBV-HLH to the doctor to provide a reference for the doctor's diagnostic process.

[0128] In the comparative examples, miRNA-375, miRNA-148a-3p, EBV-DNA, Hb, PIT, and EBV-DNA+miRNA-375 were used as indicators, and plasma samples were tested according to the above-described embodiments. The experimental results are as follows. Figure 3 As shown, using EBV-DNA+miRNA-375 as the indicator selected in this disclosure yields the highest diagnostic efficacy. This confirms that the kit provided in this disclosure, using EBV-DNA+miRNA-375 as the diagnostic indicator, has higher accuracy.

[0129] In summary, this disclosure provides a diagnostic indicator that can specifically detect EBV-HLH and a kit that can more sensitively and accurately diagnose EBV-HLH.

[0130] Based on the foregoing, those skilled in the art will understand that the technical solutions claimed in this disclosure and their equivalents will be readily apparent. Furthermore, those skilled in the art can make appropriate modifications and alterations to the disclosed technical solutions as needed, and these modifications and improvements are also within the scope of protection of the claims in this disclosure.

Claims

1. A kit for early differential diagnosis of EBV-HLH, characterized in that, include: EBV nucleic acid detection reagent and miRNA-375 detection reagent.

2. The kit of claim 1, wherein The miRNA-375 detection reagent includes primers that can specifically bind to miRNA-375.

3. The kit of claim 2, wherein The primers include hsa-miR-375 primers; The primer sequence is: 5'-TTTGTTCGTTCGGCTCGCG-3'.

4. The kit of claim 1, wherein The EBV nucleic acid detection reagent includes: EBV nucleic acid extraction reagent, PCR reaction solution, and EBV quality control solution.

5. The kit according to claim 1, characterized in that, The miRNA-375 detection reagent also includes: plasma total RNA extraction reagent, miRNA reverse transcription reagent, and qPCR reaction reagent.

6. The reagent kit as described in claim 5, characterized in that, The plasma total RNA extraction reagent includes denaturing buffer, cel-miR-39 external reference, acid-phenol-chloroform solution, anhydrous ethanol, and elution buffer.

7. The kit according to claim 5, characterized in that, The miRNA reverse transcription reagent includes a reverse transcription mixture and reverse transcriptase.

8. The reagent kit as described in claim 5, characterized in that, The qPCR reaction reagent includes an amplification mixture.

9. The kit according to claim 1, characterized in that, The kit is applicable to test samples including plasma separated using sodium citrate anticoagulant tubes.

10. A method for assisting in the diagnosis of EBV-HLH, characterized in that, include: Obtain the EBV nucleic acid test results of the sample; Obtain the CT value of miRNA-375 in the sample; When the EBV DNA detection result is greater than 5000 copies / mL and the miRNA-375 detection CT value is less than 35, auxiliary diagnostic result information of suspected EBV-HLH is output.

Images