Application of reagent for detecting expression quantity of circular RNA hsacirc0001147 in preparation of product for diagnosing acute myelogenous leukemia of children

Abstract

The invention belongs to the technical field of biomedical detection, and particularly relates to application of a reagent for detecting the expression quantity of circular RNA hsacirc0001147 in preparation of a product for diagnosing acute myelogenous leukemia of children. The invention provides application of a reagent for detecting the expression quantity of circular RNA hsacirc0001147 in preparation of a product for diagnosing acute myelogenous leukemia of children. According to research, hsacirc0001147 is specifically and highly expressed in peripheral blood mononuclear cells of M7 type acute myelogenous leukemia children, and the expression level of hsacirc0001147 is obviously higher than that of healthy children and other subtype AML children. The discovery reveals the strong correlation between the hsacirc0001147 and the acute myelogenous leukemia of children, so that the hsacirc0001147 becomes a molecular diagnosis marker with great potential, early, rapid and accurate identification of the acute myelogenous leukemia of children can be realized by detecting the expression level of the marker, the defects of the existing MICM diagnosis process are effectively made up, and the diagnosis accuracy of the acute myelogenous leukemia of children is improved. The risk of missed diagnosis and misdiagnosis is reduced, and an important basis is provided for formulating an accurate treatment strategy clinically.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical detection, and particularly relates to application of a reagent for detecting expression of circular RNA hsa_circ_0001147 in preparation of a product for diagnosing childhood acute myeloid leukemia. BACKGROUND

[0002] Childhood acute myeloid leukemia (AML) is a common hematological malignancy in children, accounting for about 15% of childhood leukemia. FAB classification divides AML into different subtypes (M0-M7) according to its cell origin and differentiation stage. M7 type AML, i.e., acute megakaryocytic leukemia (AMKL), is a rare subtype of acute myeloid leukemia characterized by abnormal proliferation of primitive megakaryocytes. The incidence of childhood AMKL accounts for 3.0%-10.0% of childhood AML, and is often accompanied by Down syndrome. This subtype of AML usually has a poor prognosis, and early and accurate diagnosis is crucial for developing treatment strategies and improving the prognosis of children.

[0003] Currently, clinical diagnosis of AMKL often uses a combination of cell morphology, immunology, cytogenetics and molecular biology detection methods, i.e., MICM classification method. The existing diagnostic technology is limited to MICM combination. Although this method is effective, it has the disadvantages of complex process, long time consumption, high requirements for technology and samples, and insufficient diagnostic efficiency in some difficult cases. The incidence of childhood AMKL is low, and its primitive megakaryocyte morphology is variable and difficult to identify. Moreover, chemical staining has no specific markers. At the same time, AMKL is often accompanied by myelofibrosis, and dry aspiration often occurs during puncture, resulting in a bone marrow blast cell ratio of <20%, and a high rate of clinical misdiagnosis and misdiagnosis. There is currently a lack of highly sensitive and specific molecular markers for M7 type AML for differential diagnosis.

[0004] Therefore, how to use a new technology with simple operation, high sensitivity, strong specificity, and capable of effectively supplementing or even partially replacing the existing process, or a liquid biopsy marker to replace the existing detection index, to perform early differential diagnosis of childhood AMKL is a problem to be solved in the field. SUMMARY

[0005] Based on this, one embodiment of the present application provides application of a reagent for detecting expression of circular RNA hsa_circ_0001147 in preparation of a product for diagnosing childhood acute myeloid leukemia.

[0006] One aspect of the present application provides application of a reagent for detecting expression of circular RNA hsa_circ_0001147 in preparation of a product for diagnosing childhood acute myeloid leukemia.

[0007] In some embodiments, the childhood acute myeloid leukemia diagnosed by the diagnostic product includes M7 type childhood acute myeloid leukemia.

[0008] In some embodiments, the reagent for detecting the expression level of the circular RNA hsa_circ_0001147 comprises a pair of detection primers.

[0009] In some embodiments, the pair of detection primers is selected from one or more pairs of primers with nucleotide sequences as shown in SEQ ID NO. 1-4.

[0010] In some embodiments, the pair of detection primers further comprises a pair of primers for detecting an internal reference.

[0011] The pair of primers for detecting an internal reference is selected from one or more pairs of primers with nucleotide sequences as shown in SEQ ID NO. 5-8.

[0012] Another aspect of the present application provides a pair of detection primers, which is selected from one or more pairs of primers with nucleotide sequences as shown in SEQ ID NO. 1-4.

[0013] In some embodiments, the pair of detection primers further comprises one or more pairs of primers with nucleotide sequences as shown in SEQ ID NO. 5-8.

[0014] Another aspect of the present application provides a diagnostic product for childhood acute myeloid leukemia, which comprises the reagent defined above.

[0015] In some embodiments, the diagnostic product for childhood acute myeloid leukemia comprises a kit.

[0016] Another aspect of the present application provides a method for detecting the expression level of the circular RNA hsa_circ_0001147, which comprises using the detection primers or the detection kit described above to detect the expression level of the circular RNA hsa_circ_0001147 in a sample to be tested.

[0017] This application provides the application of a reagent for detecting the expression level of circular RNA hsa_circ_0001147 in the preparation of diagnostic products for childhood acute myeloid leukemia (AML). This application reveals that hsa_circ_0001147 is specifically and highly expressed in peripheral blood mononuclear cells of AML patients, with expression levels significantly higher than in healthy children and other subtypes of AML. This finding reveals a strong correlation between hsa_circ_0001147 and AML, making it a highly promising molecular diagnostic biomarker. Furthermore, by detecting the expression level of this biomarker, early, rapid, and accurate identification of childhood AML can be achieved, effectively compensating for the shortcomings of existing MICM diagnostic procedures, reducing the risk of missed and misdiagnosed cases, and providing important evidence for the development of precise clinical treatment strategies. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The identification result is for the circular RNA hsa_circ_0001147;

[0020] Figure 2 This is a schematic diagram of the structure of the circular RNA hsa_circ_0001147;

[0021] Figure 3 Results of qRT-PCR detection of circular RNA hsa_circ_0001147 expression in pediatric AML clinical samples;

[0022] Figure 4 The expression results of circular RNA hsa_circ_0001147 in various AML cell lines were detected by qRT-PCR.

[0023] Figure 5 ROC curve and corresponding area under the curve (AUC) value of peripheral blood of circular RNA hsa_circ_0001147 for the diagnosis of AMKL in children. Detailed Implementation

[0024] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0025] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0026] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0027] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0028] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0029] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0030] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0031] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0032] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0033] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0034] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0035] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0036] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0037] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0038] To address the technical challenge of lacking highly sensitive, specific, and easy-to-use molecular markers for the auxiliary differential diagnosis of childhood AML, this application provides, on the one hand, the application of a reagent for detecting the expression level of circular RNA hsa_circ_0001147 in the preparation of diagnostic products for childhood acute myeloid leukemia.

[0039] hsa_circ_0001147 is located at chr20:34304661-34313077, formed by reverse splicing of exons 7-10 of RNA-binding motif protein 39 (RBM39), with a length of 475 bp. Its transcript is NM_001242600.2. This circRNA sequence is stable, easy to detect, and suitable as a biomarker for liquid biopsy.

[0040] This application reveals that hsa_circ_0001147 is specifically and highly expressed in peripheral blood mononuclear cells (PBMCs) of children with M7 AML, with expression levels significantly higher than in healthy children and children with other subtypes of AML. This finding is the first to demonstrate the strong correlation between hsa_circ_0001147 and childhood M7 AML, making it a highly promising molecular diagnostic biomarker. Using peripheral blood PBMCs as the test sample, compared to traditional bone marrow aspiration, offers advantages such as being non-invasive or minimally invasive, easy to collect, and highly reproducible, greatly improving the convenience of testing and patient compliance. By detecting the expression level of this biomarker, early, rapid, and accurate identification of M7 AML can be achieved, effectively compensating for the shortcomings of existing MICM diagnostic procedures, reducing the risk of missed and misdiagnosed cases, and providing important evidence for the development of precise clinical treatment strategies.

[0041] In some embodiments, acute myeloid leukemia includes childhood acute myeloid leukemia.

[0042] In some embodiments, the childhood acute myeloid leukemia includes M7 type childhood acute myeloid leukemia.

[0043] In some embodiments, the reagents for detecting the expression level of the circular RNA hsa_circ_0001147 include detection primer pairs.

[0044] In some embodiments, the detection primer pairs are selected from one or more primer pairs with nucleotide sequences such as SEQ ID NO.1-SEQ ID NO.4.

[0045] hsa_circ_0001147-F 5'-GGGATATGGATTTATTACAAGAACC-3' (SEQ ID NO. 1).

[0046] hsa_circ_0001147-R 5'-TTCCTACTGTAGAGAAAAACTCTTC-3' (SEQ ID NO. 2).

[0047] hsa_circ_0001147-F 5'-AGGAATTGCTTATGTGGAGTTCGTC-3' (SEQ ID NO. 3).

[0048] hsa_circ_0001147-R 5'-TCAATAGGTTCTTCTGTGATGCCTGT-3' (SEQ ID NO. 4).

[0049] This primer pair contains primers specifically designed to amplify the hsa_circ_0001147 backsplicing site (BSJ). By designing primers specifically targeting the backsplicing site, the specificity of the detection can be ensured, effectively distinguishing circular RNA from its linear parent transcripts, avoiding cross-reactivity, and thus improving diagnostic accuracy.

[0050] This primer pair is an experimentally validated, preferred primer pair capable of efficiently and specifically amplifying the hsa_circ_0001147 backsplicing site. Using this primer pair for detection yields high signal-to-noise ratio and highly reproducible experimental results, further ensuring the reliability of the diagnosis.

[0051] In some embodiments, the detection primer pair further includes an internal reference detection primer pair; the internal reference detection primer is selected from one or more primer pairs with nucleotide sequences as shown in SEQ ID NO.4-SEQ ID NO.8. The internal reference gene GAPDH is relatively stably expressed in various tissues and cells. By simultaneously detecting GAPDH, the RNA amount, reverse transcription efficiency, and PCR amplification efficiency of the samples can be standardized and corrected, eliminating experimental errors and making the target gene expression levels comparable between different samples, thus improving the accuracy and reliability of the data.

[0052] 5'-TCTCCTCTGACTTCAACAGCGACA-3' (SEQ ID NO. 5).

[0053] 5'-CCCTGTTGCTGTAGCCAAATTCGT-3' (SEQ ID NO. 6).

[0054] 5'-CGACTTCAACAGCAACTCCCACTCTTC-3' (SEQ ID NO. 7).

[0055] 5'-TGGGTGGTCCAGGGTTTCTTACTCCTT-3' (SEQ ID NO. 8).

[0056] In another aspect, this application provides a detection primer pair, which is selected from one or more primer pairs with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.4.

[0057] In some embodiments, the detection primer pair further includes one or more pairs of primer pairs with nucleotide sequences as shown in SEQ ID NO.4-SEQ ID NO.8.

[0058] Another aspect of this application provides a detection kit comprising the aforementioned detection primer pair.

[0059] In some embodiments, the kit further includes one or both of RNA extraction reagents and reverse transcription reagents.

[0060] Another aspect of this application provides a method for detecting the expression level of circular RNA hsa_circ_0001147, comprising using the detection primer pair or the detection kit described in the detection method to detect the expression level of circular RNA hsa_circ_0001147 in the sample to be tested.

[0061] The differential diagnosis includes distinguishing M7 type childhood acute myeloid leukemia from other subtypes of childhood acute myeloid leukemia and / or healthy controls. The research data in this application show that hsa_circ_0001147 expression in M7 type AML is highly specific, and its expression level can effectively distinguish M7 type AML from other subtypes of AML such as M1, M2, M3, M4, M5, and M6, as well as healthy individuals, demonstrating excellent differential diagnostic ability.

[0062] Understandably, this test can be used for both disease diagnosis and treatment purposes and non-disease diagnosis and treatment purposes.

[0063] Specifically, the method for detecting the expression level of circular RNA hsa_circ_0001147 includes the following steps: Step 1: Isolating peripheral blood mononuclear cells (PBMCs) from the peripheral blood of the subject; Step 2: Extracting total RNA from the PBMCs; Step 3: Reverse transcribing the total RNA into cDNA; Step 4: Detecting the expression level of hsa_circ_0001147 in the cDNA using real-time quantitative PCR (qPCR). This method provides a complete and standardized operating procedure, from sample processing to final result interpretation, ensuring the standardization of the detection process and the comparability of the results. Based on qPCR technology, this method has the advantages of high sensitivity, strong specificity, accurate quantification, and high throughput, and can meet the clinical requirements for molecular marker detection.

[0064] In some embodiments, the qPCR reaction procedure in step 4 includes: pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 5 seconds, annealing / extension at 60°C for 30 seconds, repeated for 40 cycles. This reaction procedure is an optimized qPCR amplification condition suitable for the primer pairs of this application, ensuring amplification efficiency and specificity, and obtaining stable amplification curves and accurate CT values.

[0065] In some embodiments, the total RNA extraction method in step 2 includes Trizol lysis, chloroform extraction, isopropanol precipitation, and ethanol washing. The Trizol method is a classic RNA extraction method that effectively lyses cells, inhibits RNase activity, and separates high-quality total RNA from proteins and DNA. RNA extracted using this method has high purity and good integrity, providing a high-quality template for subsequent reverse transcription and qPCR detection, making it a preferred method to ensure successful detection.

[0066] In some embodiments, the method further includes step 5: plotting receiver operating characteristic (ROC) curves based on the expression levels of hsa_circ_0001147 and calculating the area under the curve (AUC) value to evaluate its efficacy in the differential diagnosis of M7 type acute myeloid leukemia in children. ROC curve analysis is the internationally recognized gold standard for evaluating the efficacy of diagnostic tests. By calculating the AUC value, the sensitivity and specificity of hsa_circ_0001147 as a diagnostic biomarker can be objectively and quantitatively evaluated, providing strong statistical evidence for clinical application. The results of this application show that the AUC value of hsa_circ_0001147 is as high as 0.96, indicating that it has extremely high diagnostic value.

[0067] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0068] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0069] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0070] Example 1

[0071] I. Genomic localization and transcript annotation

[0072] The location and transcript information of circRNA were determined using the online software CircInteractome (https: / / circinteractome.nia.nih.gov / ) and circBase query.

[0073] The online software CircInteractome (https: / / circinteractome.nia.nih.gov / ) predicted that exons 7 to 10 of RBM39 could be reverse spliced ​​to form a circular RNA (circRNA) hsa_circ_0001147, which is 475 bp long (as shown in Table 1-1-Table 1-2). By querying RBM39 through circBase, the transcript of hsa_circ_0001147 was determined to be NM_001242600.2 (as shown in Table 2-1-Table 2-2).

[0074] Table 1-1

[0075]

[0076] Table 1-2

[0077]

[0078] Table 2-1

[0079]

[0080] Table 2-2

[0081]

[0082] II. Verification of the circular structure of circRNA

[0083] 1. RNA extraction from peripheral blood PBMCs

[0084] (1) Extraction of peripheral blood PBMCs

[0085] A. Centrifuge the blood collection tube at 2000 rpm for 10 minutes, and resuspend the lower red substance in PBS at a 1:1 volume ratio. After mixing, slowly transfer the mixture along the tube wall into another centrifuge tube containing 3 mL of lymphocyte separation medium, ensuring the mixture is above the surface of the lymphocyte separation medium. Centrifuge at 1500 rpm for 20 minutes at 22°C.

[0086] B. After centrifugation, the liquid surface is divided into 3 layers. The first layer is a white flocculent layer, which is the PBMC layer. Carefully aspirate the PBMC layer and transfer it into a centrifuge tube containing 4 mL of PBS. Mix well and centrifuge at 4°C and 1800 rpm for 10 minutes.

[0087] C. Discard the supernatant, add 2 mL of red blood cell lysis buffer to the cell pellet, mix well by pipetting for 2 minutes, then add 2 mL of PBS, mix well, and centrifuge at 1800 rpm and 4°C for 10 minutes.

[0088] D. Discard the supernatant, add 1 mL of PBS to the cell pellet for washing, mix well by pipetting, transfer to a 1.5 mL centrifuge tube, centrifuge at 2500 rpm and 4°C for 5 minutes, then discard the supernatant and retain the cell pellet.

[0089] (2) PBMC RNA extraction

[0090] A. Add 1 mL of Trizol to the collected PBMCs, and pipette until the lysis buffer is clear and free of obvious precipitate, then let it stand at room temperature for 5 minutes.

[0091] B. Add 1 / 5 volume of chloroform to the above lysis buffer, mix thoroughly, and let stand at room temperature for 5 minutes.

[0092] C. Centrifuge at 4℃, 12000g for 15 minutes. Carefully remove the centrifuge tube. At this point, the homogenate will be divided into three layers: the supernatant (containing RNA), the intermediate protein layer, and the lower organic phase. Transfer the supernatant to another new centrifuge tube.

[0093] D. Add 1 / 2 volume of isopropanol to the supernatant, mix thoroughly, let stand at room temperature for 10 minutes, centrifuge at 12000g and 4℃ for 10 minutes, and discard the supernatant after centrifugation.

[0094] E. Add an equal volume of 80% ethanol (pre-cooled to -20℃) to the centrifuge tube to wash the RNA precipitate and the walls of the centrifuge tube. Centrifuge at 7500g, 4℃ for 5 minutes. Carefully discard the supernatant, being careful not to touch the precipitate.

[0095] F. Open the centrifuge tube cap, let the precipitate dry at room temperature for about 5 minutes, then add an appropriate amount of RNase-free water to the centrifuge tube to dissolve the RNA.

[0096] (3) Determine the purity and concentration of RNA

[0097] A. Clean the Thermo Fisher microplate and add the sample to be tested. Place it in the microplate reader and select "DNA / RNA Nucleic Acid Concentration Detection".

[0098] B. Record the concentration, 260 / 280 ratio, and 260 / 230 ratio of the detected RNA. The OD260 / 280 ratio is used to assess whether there is RNA degradation or protein contamination in the extracted RNA, and the OD260 / 230 ratio is used to assess whether the extracted RNA is contaminated by organic matter or salt ions.

[0099] 2. Peripheral blood PBMC RNA is reverse transcribed into cDNA

[0100] (1) Genomic DNA removal reaction

[0101] Take 1 μg of extracted Total RNA, add 2 μL of 5×gDNA Eraser Buffer and 1 μL of gDNA Eraser, and add RNase-free water to make the total reaction volume 10 μL. Set the PCR instrument to 42℃ and react for 2 minutes to remove gDNA.

[0102] (2) Reverse transcription into cDNA

[0103] A. Add 1 μL of Prime Script RT Enzyme Mix, 1 μL of RT Primer Mix, 4 μL of 5× Prime Script Buffer, and 4 μL of RNase-free water to 10 μL of the reaction solution containing gDNA, and mix gently until homogeneous.

[0104] B. The PCR instrument was set to the following reaction conditions: 37℃ for 15 minutes, 85℃ for 5 seconds, and stored at 4℃. Reverse transcription was then started to obtain cDNA.

[0105] 3. Extraction of gDNA from peripheral blood PBMCs

[0106] A. Add 20 μL of PBS to the collected PBMCs for resuspending, then add 18 μL of Buffer GA and vortex to suspend.

[0107] B. Add 20 μL of proteinase K solution to the tube, mix thoroughly, then add 200 μL of Buffer GB, shake for 15 seconds, centrifuge quickly to remove water droplets from the tube wall, and let stand at 70°C for 10 minutes until the solution becomes clear.

[0108] C. Add 200 μL of anhydrous ethanol, shake thoroughly for 15 seconds, and then centrifuge quickly to remove water droplets from the tube wall.

[0109] D. Add the above solution to the adsorption column CB3, place the adsorption column in the collection tube, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0110] E. Add 500 μL of Buffer GD to the adsorption column CB3, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0111] F. Add 600 μL of wash buffer PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0112] G. Repeat step E.

[0113] H. Place the adsorption column back into the collection tube, centrifuge at 12000 rpm for 2 minutes, discard the waste liquid, and let the adsorption column dry completely at room temperature for about 5 minutes.

[0114] 1. Transfer the adsorption column to a new centrifuge tube, add 50 μL of elution buffer TE, incubate at room temperature for 5 minutes, then centrifuge at 12000 rpm for 2 minutes to collect the solution into the centrifuge tube.

[0115] 4. Design primers for amplification

[0116] Using peripheral blood mononuclear cells (PBMCs) cDNA and gDNA from children with AML as templates, two pairs of primers were designed for each target circRNA and internal control GAPDH. Diverging primers were used to specifically amplify the back splice site (BSJ) of the circular RNA, while converging primers were used to amplify the linear transcript. The PCR instrument was set to pre-denaturation: 95°C, 30 seconds (1 cycle); denaturation: 95°C, 5 seconds (40 cycles); annealing / extension: 60°C, 30 seconds (40 cycles), and PCR amplification was performed using the aforementioned primers.

[0117] 5. Perform agarose gel electrophoresis on the above PCR products and determine the results.

[0118] A. Assemble the gel casting apparatus. Place the gel casting glass onto the gel casting plate, then insert the gel casting comb. Weigh 0.2g of agarose, add 20mL of TAE buffer, and microwave on high for 1 minute. After the agarose is completely dissolved, cool to about 55℃ (a temperature that is not hot to the touch). Add 2uL of nucleic acid dye at a ratio of 1:10000, gently shake to mix evenly, and carefully pour into the gel casting plate. Let it stand at room temperature for 30 minutes until it solidifies.

[0119] B. Pull the gel casting comb upwards, place the gel into the electrophoresis tank, add TAE buffer until the liquid surface covers the gel, add 10uL of DNA sample into the sample well, set the electrophoresis tank to 150V, and perform gel electrophoresis for 20 minutes.

[0120] C. After electrophoresis, observe the bands under ultraviolet light.

[0121] 6. Perform Sanger sequencing on the cDNA obtained from PCR amplification.

[0122] The results show:

[0123] (1) PCR and agarose gel electrophoresis were used to identify the presence of hsa_circ_0001147 in pediatric AML cells.

[0124] Two pairs of primers were designed targeting hsa_circ_0001147 (primer sequences are detailed in Table 3). Using cDNA and gDNA from pediatric AML cells as templates, PCR amplification was performed using the two primer pairs. The PCR products were then subjected to agarose gel electrophoresis. The banding results showed that the divergent primers could amplify hsa_circ_0001147 from cDNA, while the polymeric primers could amplify hsa_circ_0001147 and the internal control GAPDH from cDNA and gDNA, respectively (primer sequences are detailed in Table 3), proving that hsa_circ_0001147 does indeed exist in cells (e.g., hsa_circ_0001147 is present in cells). Figure 1 (As shown).

[0125] Table 3: Primer sequences

[0126]

[0127] (2) Sanger sequencing

[0128] Sanger sequencing of the cDNA revealed that the backsplicing site (BSJ) of hsa_circ_0001147 was ACA│AGA (e.g., Figure 2 (As shown).

[0129] III. qPCR detection of circRNA expression in peripheral blood PBMCs of children with AML

[0130] A. Prepare the reaction system in a 96-well qPCR plate. Add 1 μL of cDNA, 1 μL of primer, 5 μL of 2×SYBRqPCR mix, and 3 μL of RNase-free water to each well. Set up 3 replicates for each sample and set up template-free negative control wells at the same time.

[0131] B. Run the following program on a real-time quantitative PCR instrument: Pre-denaturation: 95°C, 30 seconds (1 cycle); Denaturation: 95°C, 5 seconds (40 cycles); Annealing / Extension: 60°C, 30 seconds (40 cycles).

[0132] C. Calculate and analyze the results based on the CT values.

[0133] Specifically, this included collecting peripheral blood samples from children with different subtypes of AML, including 1 case of M1, 9 cases of M2, 2 cases of M3, 6 cases of M4, 9 cases of M5, 1 case of M6, 4 cases of M7, and 4 healthy controls. After extracting peripheral blood PBMCs, the expression of hsa_circ_0001147 in the clinical samples of children with AML was detected by RT-qPCR. The results showed that the expression of hsa_circ_0001147 in the PBMCs of children with M7 AML was significantly higher than that in the healthy control group and other AML subtype groups (e.g., ...). Figure 3 (As shown).

[0134] IV. Detection of circRNA expression in different AML subtype cell lines

[0135] (1) RNA extraction from AML cells

[0136] A. Culture each type of AML cell line to the logarithmic growth phase, and collect approximately 1×10⁻⁶ cells from each AML type by centrifugation. 6 Collect cells, discard the supernatant culture medium, add 1 mL of Trizol to the collected cells, and pipette until the lysis buffer is clear and there is no obvious precipitate, then let stand at room temperature for 5 minutes.

[0137] B. Add 1 / 5 volume of chloroform to the above lysis buffer, mix thoroughly, and let stand at room temperature for 5 minutes.

[0138] C. Centrifuge at 4℃, 12000g for 15 minutes. Carefully remove the centrifuge tube. At this point, the homogenate will be divided into three layers: the supernatant (containing RNA), the intermediate protein layer, and the lower organic phase. Transfer the supernatant to another new centrifuge tube.

[0139] D. Add 1 / 2 volume of isopropanol to the supernatant, mix thoroughly, let stand at room temperature for 10 minutes, centrifuge at 12000g and 4℃ for 10 minutes, and discard the supernatant after centrifugation.

[0140] E. Add an equal volume of 80% ethanol (pre-cooled to -20℃) to the centrifuge tube to wash the RNA precipitate and the walls of the centrifuge tube. Centrifuge at 7500g, 4℃ for 5 minutes. Carefully discard the supernatant, being careful not to touch the precipitate.

[0141] F. Open the centrifuge tube cap, let the precipitate dry at room temperature for about 5 minutes, then add an appropriate amount of RNase-free water to the centrifuge tube to dissolve the RNA.

[0142] (2) Determine the purity and concentration of RNA

[0143] A. Clean the Thermo Fisher microplate and add the sample to be tested. Place it in the microplate reader and select "DNA / RNA Nucleic Acid Concentration Detection".

[0144] B. Record the concentration, 260 / 230 ratio, and 260 / 280 ratio of the detected RNA. The OD260 / 280 ratio is used to assess whether there is RNA degradation or protein contamination in the extracted RNA. The OD260 / 230 ratio is used to assess whether the extracted RNA is contaminated by organic matter or salt ions.

[0145] (3) Genomic DNA removal reaction

[0146] Take 1 μg of extracted Total RNA, add 2 μL of 5×gDNA Eraser Buffer and 1 μL of gDNA Eraser, and add RNase-free water to make the total reaction volume 10 μL. Set the PCR instrument to 42℃ and react for 2 minutes to remove gDNA.

[0147] (4) Reverse transcription into cDNA

[0148] A. Add 1 μL of Prime Script RT Enzyme Mix, 1 μL of RT Primer Mix, 4 μL of 5× Prime Script Buffer, and 4 μL of RNase-free water to 10 μL of the reaction solution containing gDNA, and mix gently until homogeneous.

[0149] B. Set the PCR instrument reaction conditions to 37℃ for 15 minutes, 85℃ for 5 seconds, and store at 4℃, then begin reverse transcription to obtain cDNA.

[0150] (5) qPCR detection of circRNA expression

[0151] A. Prepare the reaction system in a 96-well qPCR plate. Add 1 μL of cDNA, 1 μL of primer, 5 μL of 2×SYBRqPCR mix, and 3 μL of RNase-free water to each well. Set up 3 replicates for each sample and set up template-free negative control wells at the same time.

[0152] B. Run the following program on a real-time quantitative PCR instrument: Pre-denaturation: 95°C, 30 seconds (1 cycle); Denaturation: 95°C, 5 seconds (40 cycles); Annealing / Extension: 60°C, 30 seconds (40 cycles).

[0153] C. Calculate and analyze the results based on the CT values.

[0154] Specifically, this includes: extracting RNA from AML cells SKNO-1 (AML-M2), OCI-AML-3 (AML-M4), NB4 (AML-M3), Mono-Mac-6 (AML-M5), and M07e (AML-M7), and detecting the expression of hsa_circ_0001147 in each AML subtype cell line by RT-qPCR.

[0155] The results showed that hsa_circ_0001147 expression in the M7 AML cell line M07e was significantly higher than in other subtype AML cell lines (e.g., ...). Figure 4 (As shown).

[0156] V. ROC Curve Plotting and Result Interpretation

[0157] Receiver operating characteristic (ROC) curves were plotted using GraphPad Prism 9.5 software, and the corresponding area under the curve (AUC) was calculated to assess diagnostic efficacy. Normally distributed continuous data were expressed as mean ± standard deviation (s), and unpaired Student's t-tests were used for comparisons between groups. Count data were expressed as cases or rates, and chi-square tests were used for comparisons between groups. 2 Test. A p-value < 0.05 is considered statistically significant.

[0158] ROC curve interpretation: The closer the curve is to the upper left corner, the better the diagnostic performance; the diagonal line represents random guessing with no diagnostic value. AUC value interpretation: 0.5-0.7: low diagnostic value; 0.7-0.9: moderate diagnostic value; ≥0.9: high diagnostic value.

[0159] GraphPad Prism 9.5 software was used to plot receiver operating characteristic (ROC) curves and calculate the corresponding area under the curve (AUC) to assess diagnostic efficacy.

[0160] The results showed that the AUC of hsa_circ_0001147 in diagnosing AMKL was 0.96 (P=0.003). The ROC curve of hsa_circ_0001147 was close to the upper left corner, and the corresponding area under the curve (AUC) was ≥0.9, demonstrating its extremely high predictive diagnostic value (e.g., Figure 5 (As shown).

[0161] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. Application of reagents for detecting the expression level of circular RNA hsa_circ_0001147 in the preparation of diagnostic products for childhood acute myeloid leukemia.

2. The application according to claim 1, characterized in that, The diagnostic product diagnoses childhood acute myeloid leukemia, including M7 type childhood acute myeloid leukemia.

3. The application according to claim 1, characterized in that, The reagents for detecting the expression level of circular RNA hsa_circ_0001147 include the detection primer pair.

4. The application according to claim 3, characterized in that, The detection primer pairs are selected from one or more primer pairs with nucleotide sequences such as SEQ ID NO.1-SEQ ID NO.

4.

5. The application according to claim 4, characterized in that, The detection primer pair also includes an internal reference detection primer pair; The internal reference detection primers are selected from one or more primer pairs with nucleotide sequences as shown in SEQ ID NO.5-SEQ ID NO.

8.

6. Detection primer pair, characterized in that, The detection primer pairs are selected from one or more primer pairs with nucleotide sequences such as SEQ ID NO.1-SEQ ID NO.

4.

7. The detection primer pair according to claim 6, characterized in that, The detection primer pairs also include one or more of the primer pairs with nucleotide sequences as shown in SEQ ID NO.5-SEQ ID NO.

8.

8. A diagnostic product for acute myeloid leukemia in children, characterized in that, Including the reagents defined in claims 1 to 5.

9. The diagnostic product for acute myeloid leukemia in children according to claim 8, characterized in that, The diagnostic product for childhood acute myeloid leukemia includes a reagent kit.

10. A method for detecting the expression level of circular RNA hsa_circ_0001147, characterized in that it includes using the detection primer pair described in any one of claims 6 to 7 or the diagnostic product for acute myeloid leukemia in children as described in any one of claims 8 to 9 to detect the expression level of circular RNA hsa_circ_0001147 in the sample to be tested.

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