Integrated RNA absolute quantitative analysis system for sepsis and sepsis and acute kidney injury risk stratification and application thereof

Abstract

The invention discloses an integrated RNA (Ribonucleic Acid) absolute quantitative analysis system for risk stratification of sepsis and related renal injury and application of the integrated RNA absolute quantitative analysis system. The core of the system is a customized pseudovirus standard substance, and a genome of the pseudovirus standard substance contains a marker TCONS00016233 sequence; an MS2 phage pseudovirus is adopted as an RNA standard substance for absolute quantification, the copy number of a biomarker TCONS00016233 is obtained, and risk level information is output based on two specific thresholds (22 copy / reaction and 49 copy / reaction) which are clinically verified; the pseudovirus standard substance and the specific biomarker are combined with an integrated quantitative detection technology, so that the risk stratification accuracy is remarkably improved, reliable technical support is provided for the risk stratification of sepsis and related renal injury thereof, the special detection product is high in adaptability, the system is convenient to operate, and the kit has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an integrated RNA absolute quantification analysis system for sepsis and sepsis with acute kidney injury risk stratification, and also relating to the application of this system. Background Technology

[0002] Sepsis is a common critical illness in clinical practice. It is also a frequent cause of acute kidney injury (AKI) in critically ill patients. Currently, indicators commonly used for sepsis diagnosis include procalcitonin, heparin-binding protein, and C-reactive protein, but these indicators have low specificity in detecting sepsis. The diagnosis of AKI currently relies mainly on serum creatinine and urine output, but these are affected by various factors and cannot accurately reflect changes in renal function in a timely manner, lacking sufficient sensitivity and specificity for AKI diagnosis. Patent CN107541564 A discloses the application of the molecular marker TCONS_00016233 in the early diagnosis and prediction of sepsis complicated by acute kidney injury. The detection method involves conventional PCR combined with agarose gel electrophoresis. The electrophoresis images are scanned using an image analysis system to obtain grayscale values ​​IA (optical density scan values), which represent the brightness of the target band on the gel, i.e., the amount of the target band. The IA ratio of the target fragment to the internal control GAPDH represents the relative amount of PCR product. This method is a semi-quantitative method, and a relatively quantitative one at that. It suffers from drawbacks such as cumbersome operation, low throughput, inability to accurately quantify, and strong subjectivity of results. Currently, plasmid DNA is commonly used as a standard for quantification of this marker. However, plasmid standards have a fundamental technical defect: they cannot simulate the RNA extraction and reverse transcription process in clinical samples, leading to unmonitored systematic biases in the quantitative results.

[0003] Furthermore, while existing quantitative real-time PCR (qPCR) detection technology is widely used for nucleic acid quantification, and RNA pseudovirus standards based on MS2 bacteriophage and other sources are available, current solutions primarily focus on the quantitative technical aspects themselves, lacking deep integration with specific disease risk stratification models. Specifically, how to combine the absolute quantitative data obtained from RNA pseudovirus standards based on MS2 bacteriophage and other sources with large-sample clinically validated cutoff values ​​that effectively distinguish between "sepsis risk" and "sepsis with acute kidney injury risk" to form a standardized, integrated detection and risk assessment system remains a pressing technical challenge.

[0004] Therefore, there is an urgent need in this field for a new technical solution that can overcome the blind spots of traditional quantitative methods in process monitoring and seamlessly integrate highly accurate absolute quantitative results with validated clinical risk stratification rules, thereby providing a more accurate and reliable integrated tool for risk assessment of sepsis and sepsis with acute kidney injury. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings of existing technologies and provide an integrated RNA absolute quantification analysis system for risk stratification of sepsis and sepsis with acute kidney injury, as well as its applications. This system enables end-to-end monitoring from sample processing to final detection, obtaining accurate, stable, and reproducible absolute quantification results. Furthermore, it clarifies for the first time its optimal threshold for risk stratification of sepsis and sepsis with acute kidney injury, and demonstrates rigorously validated superior performance, providing a reliable tool for precision clinical management.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] An integrated RNA absolute quantification analysis system for risk stratification of sepsis and related kidney injury includes an RNA pseudovirus standard, wherein the RNA pseudovirus standard is MS2 bacteriophage pseudovirus, the genome of which contains the nucleic acid sequence shown in SEQ ID NO:1, and its concentration is absolutely calibrated by digital PCR; the digital PCR uses specific primers and probes with sequences shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0008] Furthermore,

[0009] The analytical system also includes a silica membrane nucleic acid purification column for extracting total RNA from the sample and a lysis buffer containing phenol and guanidine isothiocyanate.

[0010] The analytical system also includes a preservation solution for MS2 phage pseudovirus standards.

[0011] The MS2 phage pseudovirus standard is suspended in a specific viral nucleic acid preservation solution, preferably Ningbo Aijiekangning Biotechnology Co., Ltd., NA-005-L.

[0012] The analytical system also includes reverse transcription reagents and real-time PCR reaction reagents.

[0013] The analytical system described above uses a series of MS2 phage pseudovirus standards of known concentrations to establish a standard curve for absolute quantification.

[0014] The analytical system, based on the standard curve, absolutely quantifies the copy number of the biomarker TCONS_00016233 in the sample to be tested; compares the copy number with a first threshold and a second threshold, wherein the second threshold is higher than the first threshold; and performs risk stratification based on the comparison results: when the copy number is greater than or equal to the first threshold but less than the second threshold, it is assessed as an increased risk of sepsis; when the copy number is greater than or equal to the second threshold, it is assessed as an increased risk of sepsis and acute kidney injury.

[0015] The analytical system has a first threshold of 22 copies / test reaction system and a second threshold of 49 copies / test reaction system.

[0016] The present invention also provides an application of the aforementioned analysis system to improve the accuracy of precise risk stratification of sepsis and sepsis with acute kidney injury.

[0017] The application must be performed within an integrated RNA absolute quantification system, and includes the following steps:

[0018] (a) Provide RNA pseudovirus standards, and use a series of standards at known concentrations to simultaneously perform real-time quantitative PCR amplification with the reverse transcription products of the test sample to establish a standard curve;

[0019] (b) Based on the standard curve, absolutely quantify the copy number of biomarker TCONS_00016233 in the test sample;

[0020] (c) Compare the copy number with a first threshold and a second threshold, wherein the second threshold is higher than the first threshold;

[0021] (d) Risk stratification based on comparison results: when the copy number is greater than or equal to the first threshold but less than the second threshold, the risk of sepsis is assessed as increased; when the copy number is greater than or equal to the second threshold, the risk of sepsis and acute kidney injury is assessed as increased.

[0022] Furthermore, the judgment rules include:

[0023] When the amplification curve of the internal reference gene GAPDH is S-shaped and the Ct value is within the effective range, and the copy number of TCONS_00016233 is lower than the first threshold, the output indicates a low risk of sepsis.

[0024] When the copy number of TCONS_00016233 is greater than or equal to the first threshold but less than the second threshold, the output indicates an increased risk of sepsis.

[0025] When the copy number of TCONS_00016233 is greater than or equal to the second threshold, the output indicates an increased risk of sepsis and acute kidney injury.

[0026] The fluorescence quantitative PCR detection module includes a fluorescence quantitative PCR instrument.

[0027] This invention excludes applications where the direct purpose is disease diagnosis or treatment.

[0028] Compared with existing technologies, the integrated RNA absolute quantification analysis system and application for risk stratification of sepsis and related kidney injury provided by this invention have the following significant advantages:

[0029] 1. This invention fundamentally improves the accuracy and reliability of absolute quantification: The core of this invention uses MS2 bacteriophage pseudovirus as an RNA standard. This standard can completely simulate the entire process of RNA extraction, reverse transcription, and PCR amplification of the target RNA (TCONS_00016233) in the sample. This fundamentally overcomes the systematic errors caused by the inability of traditional DNA standards (plasmids) to monitor steps such as reverse transcription, resulting in a final biomarker copy number that more closely approximates its true expression level, laying a precise data foundation for subsequent risk assessment.

[0030] 2. Clear clinical value: For the first time, it provides absolute quantitative cutoff values ​​(22 copies / response, 49 copies / response) for risk assessment of sepsis and sepsis with acute kidney injury, which have been validated by a large sample. The system has excellent risk stratification ability, and its area under the receiver operating characteristic curve (AUC) can reach more than 0.9. It has high sensitivity and specificity, excellent performance, and clear clinical guidance value.

[0031] 3. System stability: By using the Trizol-column combined method for RNA extraction and optimizing the preservation solution, the intra-batch coefficient of variation (CV) was controlled below 5%, ensuring the high stability and repeatability of the detection system.

[0032] 4. Provides a complete industrialization solution: This invention is not an improvement on a single component, but provides a complete standardized solution from core standards, special reagents, standard operating procedures to final interpretation rules, which greatly reduces the application threshold and makes it easy to achieve quality control and large-scale promotion.

[0033] This invention achieves accurate quantitative detection of the molecular marker TCONS_00016233 and reliable risk assessment of sepsis and sepsis with acute kidney injury through innovative pseudovirus standard design, optimized detection process and integrated analysis system. Attached Figure Description

[0034] Figure 1Agarose gel electrophoresis image for detecting TCONS_00016233;

[0035] Figure 2 Standard curve plot used to verify the linear relationship of pseudovirus standards;

[0036] Figure 3 Standard curve plot used to verify the linear relationship of pseudovirus plasma matrix;

[0037] Figure 4 The low-temperature storage stability of pseudoviruses diluted with a special preservation solution;

[0038] Figure 5 The image shows the qPCR results after pseudovirus extraction using the Trizol precipitation method.

[0039] Figure 6 The image shows the qPCR results after pseudovirus detection using the Trizol-column combined method.

[0040] Figure 7 The standard curve prepared for this invention;

[0041] Figure 8 ROC curve of sepsis test results;

[0042] Figure 9 ROC curve of the test results for sepsis complicated with acute kidney injury. Detailed Implementation

[0043] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0044] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0045] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0046] This invention proposes an integrated RNA absolute quantification analysis system and application for sepsis and sepsis with acute kidney injury risk stratification, which will be described in detail below.

[0047] Example 1: Customization, concentration determination and preparation of pseudovirus standards

[0048] 1. Design and commissioned synthesis of standard products

[0049] 1.1 Sequence Selection and Construct Preparation

[0050] The known full-length sequence of the molecular marker TCONS_00016233 (as shown in SEQ ID NO: 1) was directly selected and inserted completely into the multiple cloning site of the MS2 phage standard vector. This construction method maintains the integrity of the target sequence, requiring no codon optimization or sequence modification, ensuring its normal expression and packaging in the MS2 system.

[0051] 1.2 Outsourced Synthesis and Quality Requirements

[0052] The target sequence (SEQ ID NO: 1) was provided to Sangon Biotech (Shanghai) Co., Ltd., which was commissioned to package and produce MS2 pseudovirus particles. This invention provides specific technical specifications for the deliverables:

[0053] 1) The mature MS2 phage packaging system is required.

[0054] 2) The final product needs to be purified to ensure the removal of cell debris and unpackaged nucleic acids.

[0055] 3) Delivery confirmation: The delivered MS2 pseudovirus stock solution should contain structurally intact virus particles, and its genome must contain the complete target sequence (SEQ ID NO: 1).

[0056] 1.3 Verification

[0057] 1) Sequence accuracy: The Sanger method verifies that the inserted sequence is consistent with the reference sequence.

[0058] 2) Restriction enzyme identification: Agarose gel electrophoresis is used to detect the expected size ( Figure 1 ).

[0059] 2. Absolute quantification and traceability of standard concentrations

[0060] 2.1 Selection of Quantitative Methods

[0061] To achieve accurate and traceable quantification, this invention employs droplet digital PCR (ddPCR) technology for absolute quantification of MS2 pseudovirus stock solution. This method does not rely on a standard curve and provides copy number concentration through direct counting, ensuring the accuracy of value transfer.

[0062] 2.2 Sample Pretreatment (Simulating the Entire Detection Process)

[0063] To realistically simulate the clinical sample testing process and achieve full-process monitoring, the MS2 pseudovirus stock solution was processed as follows:

[0064] 1) Extraction: Take an appropriate amount of MS2 pseudovirus stock solution and use the same silica membrane column RNA extraction kit as the one used for clinical sample testing.

[0065] 2) Reverse transcription: The extracted RNA was synthesized into cDNA using the same reverse transcription kit as the clinical test.

[0066] 2.3 Absolute Quantitative Analysis by ddPCR

[0067] 1) Reaction composition: Take the above cDNA, add ddPCR premix (Magic-Bio, M231), specific primers (SEQ ID NO: 2 and SEQ ID NO: 3) and probe (SEQ ID NO: 4) for the target sequence (SEQ ID NO: 1), and add nuclease-free water to a total volume of 20 μL.

[0068]

[0069] 2) Droplet preparation and amplification: Place the droplets in a digital PCR instrument for droplet generation, amplification, and data acquisition and analysis. The program was set as follows: 60℃ for 5 minutes; 95℃ for 5 minutes; 45 cycles of 95℃ for 20 seconds and 60℃ for 30 seconds.

[0070] 3) Results analysis: Fluorescence signals were detected using a digital PCR instrument, and the copy number concentration (copies / μL) of the original solution was directly calculated using analysis software.

[0071] 3. Preparation and storage of standard working solutions

[0072] 3.1 Gradient dilution preparation

[0073] Based on the stock solution concentration determined by ddPCR, serial dilutions were performed using a dedicated viral nucleic acid preservation solution to prepare solutions covering six orders of magnitude (e.g., 2 × 10⁻⁶). 2 Up to 2×10 7 (copies / μL) of standard working solution.

[0074] 3.2 Packaging and Storage Conditions

[0075] The diluted standard working solution was aliquoted into small portions and stored at -20±5℃ to avoid repeated freeze-thaw cycles.

[0076] 4. Validation of Standard Products

[0077] 4.1 Verification of Linearity

[0078] A batch of standard working solutions was randomly selected, and RNA was extracted and reverse transcribed. The linearity was then verified by qPCR. A standard curve was plotted with the logarithm of concentration on the x-axis and Ct values ​​on the y-axis, requiring a correlation coefficient R² > 0.99.

[0079] The results are as follows Figure 2 As shown, R²=(-0.99785)^2=0.9957>0.99.

[0080] 4.2 Influence of matrix effect

[0081] 1) Based on the stock solution concentration determined by ddPCR, serial dilutions were performed using negative plasma to prepare solutions covering 6 orders of magnitude (2 × 10⁻⁶). 2 Up to 2×10 7 Working solution of plasma standard (copies / μL).

[0082] 2) RNA was extracted, reverse transcribed, and then its correlation was verified by qPCR. The results are as follows: Figure 3 As shown, R²=(-0.99984)^2=0.9997>0.99.

[0083] A paired t-test was performed on the amplified Ct values ​​of the standard working solution and the plasma standard working solution. The test results are as follows:

[0084]

[0085] The results showed that the Pearson correlation coefficient of 0.9987 indicated that pseudoviruses of the same dilution gradient, whether placed in preservation solution or plasma, exhibited a high correlation in their detection signals. The two-tailed P(T<=t) was 0.123>0.05, indicating that the plasma matrix had no significant impact on the detection results. The mean value of the plasma group (20.77) was slightly lower than that of the pseudovirus standard group (21.115), possibly because the target copy number in the plasma matrix was not zero, which was 0.38 copies / μL as determined by ddPCR.

[0086] Conclusion: No statistically significant plasma matrix effect was found in the current six gradient experiments. The detection signals were highly consistent in plasma and preservation solution.

[0087] 5. Technological Advantages

[0088] This preparation method has the following outstanding advantages:

[0089] Process authenticity: The standard products undergo a processing procedure that is completely identical to that of clinical samples, enabling full-process quality monitoring.

[0090] Traceability of concentration values: Absolute quantification via ddPCR ensures the accuracy and traceability of concentration values.

[0091] Ease of use: The full length of the target sequence can be used directly without complex design, which facilitates standardized production.

[0092] Stability: MS2 phage has a stable structure and, when used with a special preservation solution, ensures reliable long-term preservation.

[0093] This embodiment is the first to realize the application of MS2 pseudovirus standard to the absolute quantitative detection of this molecular marker, providing a reliable technical basis for accurate risk assessment of sepsis and acute kidney injury.

[0094] Example 2: Screening and Validation of Pseudovirus Preservative Solution (Viral Nucleic Acid Preservative Solution)

[0095] 1. Purpose of Screening

[0096] Previous studies revealed that, because the standard is an RNA pseudovirus, the MS2 pseudovirus standard diluted and preserved with conventional PBS buffer showed significant nucleic acid degradation after only two weeks of storage at -20°C (data not disclosed). To ensure the stability of the standard during transportation, storage, and use, and to guarantee the accuracy and reproducibility of the test results, this invention systematically screened and validated various commercially available virus preservation solutions.

[0097] 2. Experimental Materials and Methods

[0098] 2.1 Preservative solution to be tested

[0099] Four commercially available virus preservation solutions with different stabilization mechanisms were selected for comparison:

[0100] Preservative Solution A: Guanidine salt-based inactivating preservative solution (Brand 1)

[0101] Preservative Solution B: Inactivating preservative solution containing sodium dodecyl sarcosinate (Brand 2)

[0102] Preservative solution C: Non-inactivating preservative solution based on the Hanks buffer system (Brand 3)

[0103] Preservative solution D: Non-inactivating preservative solution containing RNA nuclease inhibitors (Ningbo Aijiekangning Biotechnology Co., Ltd., NA-005-L, containing sodium ascorbate, urea, gold tricarboxylic acid, and sodium citrate).

[0104] 2.2 Experimental Design

[0105] 1) Based on the stock solution concentration determined by ddPCR, serial dilutions were performed using four different preservation solutions to prepare solutions covering four orders of magnitude (e.g., 2 × 10⁻⁶). 2 Up to 2×10 5The standard working solution (copies / μL) was aliquoted and stored at room temperature, -20℃, and -70℃.

[0106] 2) Evaluation indicators: Performance was evaluated using the qPCR standard curve.

[0107] 3. Screening Results and Analysis

[0108] 3.1 Room temperature storage effect

[0109] The pseudovirus preserved in preservation solution A showed abnormal amplification after extraction, reverse transcription, and qPCR (the melting curve did not show the correct target product curve), indicating that preservation solution A was not compatible with the reaction system.

[0110] The CT values ​​of preservation solutions B, C, and D for qPCR detection of the target at room temperature are as follows:

[0111]

[0112]

[0113]

[0114] In Tables 4-1 and 4-2, the T-test is represented by the P-value.

[0115] The results show:

[0116] 1) At day 0, the amplification effect of preservation solution A was abnormal, indicating that the inactivating preservation solution containing guanidine salt had an impact on the detection system; the detection effects of preservation solutions B, C, and D were not significantly different from those preserved in PBS (Table 4-1).

[0117] 2) At room temperature, from 0 to 4 days, preservation solution C showed significant differences, exhibiting the fastest performance degradation. Preservation solution D showed the smallest increase in Ct value, indicating its most effective inhibition of nucleic acid degradation. For low-concentration samples, the Ct value remained almost unchanged after 4 days, demonstrating extremely strong protective ability. Preservation solution B showed significantly weaker protective ability than D (Tables 3 and 4-2).

[0118] 3.2 Low-temperature (-20℃ and -70℃) preservation effect

[0119] The pseudovirus diluted with preservation solution D was stored at -20℃ and -70℃ for 7 days, 14 days, 1 month, and 3 months, respectively, to observe its long-term stability. The results are shown in [Figure number missing]. Figure 4 .

[0120] The analysis results showed that the pseudovirus preserved in preservation solution D was frozen at -20℃ and -70℃ for up to 3 months, and its protective effect was not statistically significantly reduced compared with the 0-day sample.

[0121] 3.3 Study on the number of freeze-thaw cycles during low-temperature (-20℃ and -70℃) storage

[0122] The pseudovirus diluted with preservation solution D was stored at -20℃ and -70℃ respectively, and then subjected to freeze-thaw cycles 1, 2, and 3 times. The results showed that under both temperature conditions, the freeze-thaw cycle had no significant effect on the detection results.

[0123] 4. Conclusion

[0124] Through the systematic screening in this embodiment, the most suitable preservation solution (preservation solution D) for MS2 pseudovirus standard was successfully determined, providing a reliable guarantee for the long-term stable preservation of the standard and laying the foundation for the commercialization of the reagent.

[0125] 5. Technological Advantages

[0126] The preservation solution screening and verification method established in this embodiment has the following advantages:

[0127] Systematic evaluation: Evaluated from multiple dimensions including stability, compatibility, and functionality.

[0128] Practicality: The selected preservation solution D can effectively prevent nucleic acid degradation and ensure the quality of the standard.

[0129] Economic benefits: It extends the shelf life of standard products and reduces the frequency of replacement.

[0130] Reliability: Ensures the consistency and accuracy of test results.

[0131] Example 3: Optimization and Validation of RNA Extraction Method Adapted to Detection System

[0132] 1. Optimization Objective

[0133] This invention provides an absolute quantitative detection system for the molecular marker TCONS_00016233 based on RNA pseudovirus standards. When applying this system to clinical sample testing, the inventors discovered that using the existing Trizol / chloroform extraction-ethanol precipitation method for RNA extraction introduces uncontrollable variables, specifically:

[0134] Poor reproducibility: The precipitation step is highly dependent on the operator's experience, which leads to large fluctuations in RNA recovery rate, directly affecting the accuracy of subsequent absolute quantification and reducing the comparability of results between different operators or laboratories.

[0135] Limitations on system performance: The aforementioned instability directly leads to a decrease in the reliability of diagnostic thresholds established based on a large amount of sample data, affecting the final diagnostic accuracy (AUC) and the commercial application of the reagent kit.

[0136] Therefore, the purpose of this embodiment is to screen and verify an RNA extraction method that is perfectly compatible with the specific detection system of the present invention and ensures the overall stability and performance optimization of the system.

[0137] 2. Materials and Methods

[0138] 2.1 Experimental Materials

[0139] method:

[0140] Method 1: Traditional Trizol / chloroform extraction-isopropanol precipitation method

[0141] Method 2: RNA purification was performed after Trizol / chloroform extraction using a commercial silica membrane adsorption column (Trizol-column combined method).

[0142] Sample: Dilute the fake virus stock solution to 10 2 Up to 10 5 Order of magnitude, labeled as 1, 2, 3, 4

[0143] Reagents:

[0144] Commercially available Trizol reagent (containing a lysis buffer of phenol and guanidine isothiocyanate)

[0145] Chloroform, isopropanol, 75% ethanol (for precipitation method)

[0146] Commercially available silica membrane RNA purification column (for column chromatography).

[0147] RNase-free water

[0148] 2.2 RNA extraction (comparison of Method 1 and Method 2)

[0149] 2.2.1 The specific steps of the Trizol precipitation method are as follows:

[0150] (1) Take 25 μL of label 1, label 2, label 3 and label 4 into centrifuge tubes respectively, and add 1×PBS to each centrifuge tube to make up to 250 μL.

[0151] (2) Add 0.75 mL Trizol, and use a pipette to repeatedly aspirate and swirl until the cells are completely lysed. Let stand at room temperature (15-30℃) for 5 minutes.

[0152] (3) Add 200 μL of chloroform to the centrifuge tube, tighten the cap, mix until the solution emulsifies and turns milky white, and let stand at room temperature (15-30℃) for 5 minutes.

[0153] (4) Centrifuge at 12,000×g and 4℃ for 15 minutes. Carefully remove the centrifuge tube from the centrifuge. At this point, the homogenate is divided into three layers: a colorless supernatant (containing RNA), a white protein layer in the middle (mostly DNA), and a colored lower organic layer.

[0154] (5) Transfer the supernatant to a new centrifuge tube.

[0155] (6) Add an equal volume of isopropanol to the supernatant, invert the centrifuge tube to mix thoroughly, and let stand at room temperature for 10 minutes.

[0156] (7) Centrifuge at 12000×g and 4℃ for 10 minutes. RNA precipitate will appear at the bottom of the test tube.

[0157] (8) Discard the supernatant, add an equal amount of 75% ethanol, gently shake and wash the precipitate with Vortex, centrifuge at 7500g and 4℃ for 5 minutes, and discard the supernatant.

[0158] (9) Dry the precipitate at room temperature. After the precipitate is dried, add an appropriate amount of RNase-free water to dissolve the precipitate.

[0159] 2.2 The specific steps of the Trizol-column combination method are as follows:

[0160] (1) Take 25 μL of label 1, label 2, label 3 and label 4 into centrifuge tubes respectively, and add 1×PBS to each centrifuge tube to make up to 250 μL.

[0161] (2) Add 0.75 mL Trizol, and use a pipette to repeatedly aspirate and swirl until the cells are completely lysed. Let stand at room temperature (15-30℃) for 5 minutes.

[0162] (3) Add 200 μL of chloroform to the centrifuge tube, tighten the cap, mix until the solution emulsifies and turns milky white, and let stand at room temperature (15-30℃) for 5 minutes.

[0163] (4) Centrifuge at 12,000×g and 4℃ for 15 minutes. Carefully remove the centrifuge tube from the centrifuge. At this point, the homogenate is divided into three layers: a colorless supernatant (containing RNA), a white protein layer in the middle (mostly DNA), and a colored lower organic layer.

[0164] (5) Transfer the supernatant to a new centrifuge tube.

[0165] (6) Add 0.5 times the volume of anhydrous ethanol to the supernatant, invert the centrifuge tube to mix thoroughly, and then centrifuge.

[0166] (7) Place the adsorption column in the collection tube and transfer the mixture from step 6 above into the adsorption column. Centrifuge at 12000×g for 1 min.

[0167] (8) Discard the filtrate and reassemble the adsorption column into the collection tube. Add 500 μL of wash buffer 1 to the adsorption column. Centrifuge at 12000 × g for 30 s.

[0168] (9) Discard the filtrate and reassemble the adsorption column into the collection tube. Add 500 μL of wash buffer 2 to the adsorption column. Centrifuge at 12000 × g for 30 s.

[0169] (10) Repeat step 9.

[0170] (11) Discard the filtrate and put the adsorption column back into the collection tube. Centrifuge the empty column at 12000×g for 3 min to remove the matrix from the column.

[0171] (12) Transfer the adsorption column to a new centrifuge tube and add 20 μL of elution buffer to the center of the membrane of the adsorption column. Let stand at room temperature (15-30℃) for 2 min. Centrifuge at 12000×g for 1 min to obtain RNA sample.

[0172] 2.3 Reverse transcription and qPCR

[0173] (1) Preparation of reverse transcription system (20 μL system)

[0174]

[0175] (2) Tightly cap the PCR reaction tube, mix well, and centrifuge briefly to remove the liquid from the tube wall to the bottom. Place the PCR tube in a PCR instrument and incubate according to the table below.

[0176]

[0177] After incubation, remove the PCR reaction tube, mix well, and then incubate briefly.

[0178] (3) Preparation of qPCR reaction system (20 μL system)

[0179] 1) The qPCR reaction system for the TCONS_00016233 gene was prepared as follows:

[0180]

[0181] 2) The qPCR reaction system for the GAPDH gene was prepared as follows:

[0182]

[0183] 3) qPCR amplification program

[0184]

[0185] 4) The prepared reaction system was amplified using a fluorescence quantitative instrument.

[0186] 3. Results Analysis

[0187] When extracting pseudovirus standards using the Trizol precipitation method, RNA precipitate appears at the bottom of the test tube in step (7), which is not visible to the naked eye. Therefore, even with careful operation, stability remains a significant challenge. The results showed that the four concentration gradients were not linearly related, such as... Figure 5 The Trizol-column combined extraction method reduces the skill requirements of operators, is easier to standardize, and yields more stable results. Figure 6 .

[0188] 4. Stability of RNA extracted using the Trizol-column combined method

[0189] For fake virus standards (e.g., 2×10) 2 Up to 2×10 5 The assay was repeated 20 times using copies / μL, and the CV value of the detection Ct was calculated. The results showed that the CV value did not exceed 5%, which fully meets the precision requirements of the quantitative assay kit. The results are shown in the table below.

[0190]

[0191] 5. Technological Advantages

[0192] This method optimization has led to significant technological advancements:

[0193] Improved stability: It eliminates the subjective operational factors of the precipitation method.

[0194] Quality assurance: RNA purity is significantly improved, leading to a higher success rate in downstream applications.

[0195] Standardization: The process has been standardized, and the results are consistent for different operators.

[0196] In constructing the "Absolute Quantitative Diagnostic System Based on Pseudovirus Standards" of this invention, the inventors unexpectedly discovered that the stability of the RNA extraction step was the bottleneck restricting the performance of the entire system. Through inventive efforts, they screened and confirmed from existing technologies that the "Trizol-column combined method" was the suitable solution to solve this bottleneck and unleash the optimal performance of the system of this invention.

[0197] The Trizol-column combined method successfully overcomes the technical shortcomings of traditional precipitation methods, providing a reliable sample pretreatment scheme for the stable detection of the molecular marker TCONS_00016233, and is an important component of the detection system of this invention. This method has a high degree of standardization, reducing reliance on skilled operators, and enabling the detection system of this invention to possess the stability and robustness necessary for large-scale production and clinical application, thus overcoming a key obstacle to the industrialization of the reagent kit.

[0198] Example 4: Plotting and Establishing a Standard Curve for a Fake Virus

[0199] 1. Concentration confirmation of lncRNA pseudovirus standards

[0200] Using a customized lncRNA pseudovirus standard containing the target sequence TCONS_00016233 (see SEQ ID NO:1), the concentration of the lncRNA pseudovirus solution was found to be 2.12 × 10^6 using a third-party digital PCR platform. 7 copies / μL.

[0201] 2. Preparation of gradient standards

[0202] The lncRNA pseudovirus solution was serially diluted using the pseudovirus preservation solution validated in Example 2, yielding a standard concentration of 2.12 × 10^6. 5 copies / μL, label 2: 2.12×10^ 4 copies / μL, label 3: 2.12×10^ 3 copies / μL, label 4: 2.12×10^ 2 copies / μL.

[0203] 3. Extraction of standard products

[0204] The gradient standards were extracted using the extraction method and steps verified in Example 3.

[0205] 4. Reverse transcription of standard RNA samples

[0206] (1) Preparation of reverse transcription system (20 μL system)

[0207]

[0208] (2) Tightly cap the PCR reaction tube, mix well, and centrifuge briefly to remove the liquid from the tube wall to the bottom. Place the PCR tube in a PCR instrument and incubate according to the table below.

[0209]

[0210] After incubation, remove the PCR reaction tube, mix well, and then incubate briefly.

[0211] 5. qPCR detection

[0212] (1) Primers for qPCR amplification were designed based on the gene coding sequences of TCONS_00016233 and GAPDH. The specific primer sequences are as follows:

[0213]

[0214] (2) Prepare the qPCR reaction system according to the table below:

[0215] 1) The qPCR reaction system for the TCONS_00016233 gene was prepared as follows:

[0216]

[0217] 2) qPCR amplification program

[0218]

[0219] 3) The prepared reaction system was amplified using a SLAN96S instrument.

[0220] 4) The analysis and judgment criteria for the test results are as follows:

[0221] a. Baseline and threshold line adjustment:

[0222] The baseline adjustment principle is as follows: select a region where the fluorescence signal is relatively stable before exponential amplification; avoid signal fluctuations at the beginning of fluorescence acquisition; and reduce the endpoint (Ct) by 1-2 cycles compared to the earliest sample showing exponential amplification. Threshold setting: the threshold should be set based on its position within the exponential phase of the amplification curve.

[0223] b. Amplification requirements for labels 1 to 4:

[0224] The amplification curves of labels 1 to 4 exhibit an S-shaped amplification curve. A one-to-one correspondence is established by plotting the logarithmic concentrations of labels 1, 2, 3, and 4 in the PCR wells as the x-axis and the Ct values ​​of labels 1, 2, 3, and 4 in the PCR wells as the y-axis, and then plotting the standard curve (see [link to standard curve]). Figure 7 The relationship between the two is given by the formula y = -3.2827x + 36.491, with a correlation coefficient (r) of 1, satisfying 0.98 ≤ |r| ≤ 1, indicating that the standard curve relationship meets the experimental requirements. Subsequent clinical performance verification and analysis can be performed using this standard curve.

[0225] Example 5: Clinical Performance Validation and Analysis

[0226] 1. Sample collection

[0227] Blood samples were collected from healthy individuals (144 cases), patients with sepsis without acute kidney injury (48 cases), and patients with sepsis complicated with acute kidney injury (49 cases) using blood collection tubes containing sodium citrate anticoagulant.

[0228] Inclusion criteria for positive samples:

[0229] 1) Age: ≥ 18 years old

[0230] 2) Clinical diagnosis of sepsis without acute kidney injury, and sepsis complicated with acute kidney injury.

[0231] Inclusion criteria for healthy individuals with negative samples:

[0232] 1) Age: ≥ 18 years old;

[0233] 2) Samples of people clinically diagnosed with non-sepsis.

[0234] 2. Blood Specimen Processing

[0235] Centrifuge the collected anticoagulated whole blood at 3000 rpm for 10 minutes. The upper layer is plasma. Carefully aspirate the plasma from the top of the sample into an enzyme-free centrifuge tube.

[0236] 3. Sample extraction

[0237] 1) Take 250 μL of plasma sample and prepare RNA according to the nucleic acid extraction steps of the Trizol-column combined method in Example 3.

[0238] 2) Take 250 μL of positive control (pseudovirus) and 250 μL of blank control (nuclease-free water), and extract nucleic acid according to the Trizol-column combined method nucleic acid extraction steps in Example 3. The main purpose of the positive control and blank control is to monitor the effectiveness of the detection system in order to provide accurate and reliable test results for clinical samples.

[0239] 4. Sample reverse transcription

[0240] cDNA was prepared following the reverse transcription procedure in Example 4, using 12 μL of plasma RNA. The obtained cDNA was then analyzed by quantitative real-time PCR.

[0241] 5. qPCR detection

[0242] (1) Amplify the genes of TCONS_00016233 and GAPDH, and prepare qPCR reaction systems according to the table below.

[0243] 1) The qPCR reaction system for the TCONS_00016233 gene was prepared as follows:

[0244]

[0245] 2) The qPCR reaction system for the GAPDH gene was prepared as follows:

[0246]

[0247] 3) qPCR amplification program

[0248]

[0249] 4) The prepared reaction system was amplified using a SLAN96S instrument.

[0250] 5) The analysis and judgment criteria for the test results are as follows:

[0251] a. Baseline and threshold line adjustment:

[0252] The baseline adjustment principle is as follows: select a region where the fluorescence signal is relatively stable before exponential amplification; avoid signal fluctuations at the beginning of fluorescence acquisition; and reduce the endpoint (Ct) by 1-2 cycles compared to the earliest sample showing exponential amplification. Threshold setting: the threshold should be set based on its position within the exponential phase of the amplification curve.

[0253] b. Quality control:

[0254] ① Blank control: No amplification curve was observed for GAPDH and lncRNA TCONS_00016233 genes.

[0255] ② Positive control: The GAPDH gene amplification curve is an S-shaped amplification curve with a Ct value ≤ 23, and the lncRNA TCONS_00016233 gene amplification curve is an S-shaped amplification curve with a Ct value ≤ 30.

[0256] All of the above requirements must be met simultaneously in the same experiment; otherwise, the experiment is invalid and must be repeated.

[0257] c. Establishing the first and second thresholds

[0258] Based on the standard curve obtained in Example 4, the copy number of the positive target gene in the sample was calculated, and the first and second thresholds were determined using the ROC curve method. In addition, ROC curve analysis was performed on the detection results of 241 plasma samples (144 healthy individuals, 48 ​​cases of sepsis without acute kidney injury, and 49 cases of sepsis complicated with acute kidney injury). The results showed that a cutoff value of 22 copies was optimal for judging sepsis, and a cutoff value of 49 copies was optimal for judging sepsis complicated with acute kidney injury. Detailed analysis results are shown in [link to analysis]. Figure 8 and Figure 9 .

[0259] When the first category is defined as low risk for healthy individuals and high risk for sepsis with non-acute kidney injury and sepsis with acute kidney injury, the sensitivity is 85.6%, the specificity is 84.72%, and the area under the receiver operating curve is 0.9.

[0260] When the second category is defined as low risk for healthy individuals and those with sepsis but not acute kidney injury, and high risk for those with sepsis but complicated by acute kidney injury, the sensitivity is 91.8%, the specificity is 82.81%, and the area under the receiver operating curve is 0.928.

[0261] d. Based on the first and second thresholds, the data is established, and the specific test results of the clinical samples are interpreted as follows:

[0262] The GAPDH gene has a distinct S-shaped amplification curve and a Ct value ≤ 32; the lncRNA TCONS_00016233 gene has no amplification curve or an S-shaped amplification curve and a copy number < 22 copies, and is therefore considered a low-risk sample.

[0263] The GAPDH gene showed a distinct S-shaped amplification curve with a Ct value ≤ 32; the lncRNA TCONS_00016233 gene also showed a distinct S-shaped amplification curve.

[0264] ● When the copy number of lncRNA TCONS_00016233 is ≥22 copies, the output indicates an increased risk of sepsis, and further confirmation is recommended through sepsis diagnostic methods or other clinical diagnostic methods.

[0265] ● When the copy number of lncRNA TCONS_00016233 is ≥49 copies, the output indicates an increased risk of sepsis and acute kidney injury. Further confirmation is recommended through diagnostic methods for sepsis and acute kidney injury or other clinical diagnostic methods.

[0266] The above results demonstrate that the present invention has the advantages of high sensitivity and strong specificity, and the detection method is simple and fast, which has great application value for precise clinical management.

[0267] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An integrated RNA absolute quantification analysis system for risk stratification of sepsis and related kidney injury, characterized in that, The study includes an RNA pseudovirus standard, wherein the RNA pseudovirus standard is MS2 bacteriophage pseudovirus, whose genome contains the nucleic acid sequence shown in SEQ ID NO: 1, and whose concentration is absolutely determined by digital PCR; the digital PCR uses specific primers and probes with sequences shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

4.

2. The analysis system according to claim 1, characterized in that, It also includes a silica membrane nucleic acid purification column for extracting total RNA from samples and a lysis buffer containing phenol and guanidine isothiocyanate.

3. The analysis system according to claim 1, characterized in that, It also includes the preservation solution for MS2 phage pseudovirus standards.

4. The analysis system according to claim 1, characterized in that, It also includes reverse transcription reagents and quantitative PCR reaction reagents.

5. The analysis system according to claim 1, characterized in that, A standard curve for absolute quantification was established using a series of MS2 phage pseudovirus standards at known concentrations.

6. The analysis system according to claim 1, characterized in that, Based on the standard curve, the copy number of biomarker TCONS_00016233 in the sample to be tested is absolutely quantified; the copy number is compared with a first threshold and a second threshold, wherein the second threshold is higher than the first threshold; Risk stratification is performed based on the comparison results: when the copy number is greater than or equal to the first threshold but less than the second threshold, the risk of sepsis is assessed as increased; when the copy number is greater than or equal to the second threshold, the risk of sepsis and acute kidney injury is assessed as increased.

7. The analysis system according to claim 6, characterized in that, The first threshold is 22 copies / test reaction system, and the second threshold is 49 copies / test reaction system.

8. The application of the analysis system according to any one of claims 1-7, characterized in that, Accurate risk stratification is used to improve the accuracy of risk stratification for sepsis and sepsis with acute kidney injury.

9. The application according to claim 8, characterized in that, The application must be performed within an integrated RNA absolute quantification system, and includes the following steps: (a) Provide RNA pseudovirus standards, and use a series of standards at known concentrations to simultaneously perform real-time quantitative PCR amplification with the reverse transcription products of the test sample to establish a standard curve; (b) Based on the standard curve, absolutely quantify the copy number of biomarker TCONS_00016233 in the test sample; (c) Compare the copy number with a first threshold and a second threshold, wherein the second threshold is higher than the first threshold; (d) Risk stratification based on comparison results: when the copy number is greater than or equal to the first threshold but less than the second threshold, the risk of sepsis is assessed as increased; when the copy number is greater than or equal to the second threshold, the risk of sepsis and acute kidney injury is assessed as increased.

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