A rapid detection method for necrotizing enterocolitis based on fecal markers

Abstract

This invention discloses a rapid detection method for necrotizing enterocolitis based on fecal biomarkers. Addressing the shortcomings of existing detection methods, which focus on the inflammatory stage, have high rates of early missed diagnoses, exhibit high false-positive rates in similar immunoassays, and are costly and reliant on specialized instruments for parallel testing, this method selects fecal pH, intestinal alkaline phosphatase, and calprotectin to correspond to the three pathological stages of intestinal flora imbalance, intestinal mucosal damage, and inflammation, respectively. It constructs a two-stage, step-by-step detection process using three non-interfering detection principles: physical colorimetry, enzyme-catalyzed colorimetry, and immunochromatography. First, low-cost initial screening and sample triage are performed simultaneously using pH test strips and dry chemical colorimetry for intestinal alkaline phosphatase. Only abnormal samples from the initial screening undergo calprotectin colloidal gold confirmatory testing. A three-level semi-quantitative scoring system integrates the three results to classify low, medium, and high risk levels.

Description

Technical Field

[0001] This invention relates to the field of medical in vitro detection technology, specifically a rapid detection method for necrotizing enterocolitis based on fecal markers. Background Technology

[0002] Necrotizing enterocolitis (NUC) is a serious gastrointestinal emergency prevalent in newborns, especially in the neonatal population. It is characterized by insidious onset, rapid progression, and high mortality. Fecal biomarker detection is currently an important non-invasive method for early identification of this disease. Its core principle is to detect the levels of metabolites, functional enzymes, or inflammatory proteins in stool that are related to intestinal pathological changes, assisting clinicians in assessing the degree of intestinal damage and inflammation. Existing rapid fecal biomarker detection methods primarily utilize colloidal gold immunochromatography, typically selecting inflammatory proteins such as fecal calprotectin and lactoferrin as biomarkers. Colorimetric interpretation is achieved through specific antigen-antibody binding. Some products integrate multiple similar inflammatory biomarkers into a single test card to form a combined detection mode, enhancing the ability to identify NUC.

[0003] However, existing detection methods have significant shortcomings in practical applications. On the one hand, existing biomarkers are mostly concentrated in the inflammatory response stage, which can only reflect the inflammatory cascade state after disease progression. They cannot capture pathological changes in subclinical stages such as dysbiosis and early damage to the intestinal mucosa, resulting in a significant diagnostic window lag and making it easy to miss early cases. On the other hand, existing multi-indicator joint detection mostly uses the same immunochromatographic principle, and the interfering factors of different indicators are highly overlapping, making it difficult to effectively reduce the false positive rate through joint detection. Moreover, the parallel detection mode requires all indicators to be tested simultaneously on all samples, which is costly and cannot be used as a daily routine monitoring method in neonatal wards. In addition, some quantitative detections require special reading instruments, making the operation process complex and difficult to popularize in primary medical institutions and bedside transport scenarios. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a rapid detection method for necrotizing enterocolitis based on fecal biomarkers. This method addresses the technical problems of existing rapid fecal detection methods for necrotizing enterocolitis, such as a delayed diagnostic window period, high early missed diagnosis rate, high false positive rate of combined testing based on similar principles, high cost of parallel testing, reliance on instruments, and difficulty in bedside and grassroots application.

[0005] To address the aforementioned technical problems, this invention provides the following technical solution: a rapid detection method for necrotizing enterocolitis based on fecal biomarkers, comprising the following steps:

[0006] S1 Sample Preparation: Take fresh fecal samples from outside the body, add physiological saline and mix well to prepare fecal homogenate;

[0007] S2 First Stage Dual-Indicator Initial Screening: Take the fecal homogenate and simultaneously carry out two independent detection methods: The first method uses the precision pH test paper colorimetric method to detect the pH value of the feces, and the second method uses the enzyme-catalyzed substrate colorimetric method to detect the activity of fecal intestinal alkaline phosphatase.

[0008] S3 Initial Screening Triage Determination: The results of the two initial screening tests are interpreted separately. If both results are within the normal range, the patient is determined to be at low risk for necrotizing enterocolitis, and subsequent testing is terminated. If at least one result is within the abnormal range, the patient is determined to be a suspected case and proceeds to the second stage of confirmatory testing.

[0009] S4 Second Stage Inflammation Confirmation: For fecal homogenates of suspected cases, the content of fecal calprotectin was detected by colloidal gold immunochromatography.

[0010] S5 Comprehensive Risk Rating: Integrates the results of three tests—fecal pH, intestinal alkaline phosphatase activity, and calprotectin content—and quantifies them according to a pre-set semi-quantitative scoring system. Based on the total score, it outputs the risk level of necrotizing enterocolitis.

[0011] Further, in step S1, the mass volume concentration of the fecal homogenate is 8%~12%. After mixing, it is allowed to stand or centrifuged at a low speed of less than 3000r / min for 1~2 minutes, and the upper layer of homogenate is taken for subsequent testing.

[0012] Furthermore, in step S2, the precision pH test strip has an accuracy of 0.1 pH units. During testing, the fecal homogenate is dropped onto the reaction area of ​​the pH test strip, and the reading is completed by comparing it with the standard colorimetric card within 30 to 60 seconds; where pH 5.5 is the normal range, and pH < 5.5 is the abnormal range.

[0013] Further, in step S2, the enzymatic substrate colorimetric method uses dry chemical filter paper pre-coated with p-nitrophenyl phosphate substrate. During detection, 1-2 drops of fecal homogenate are added to the reaction area of ​​the filter paper. After reacting at room temperature for 3-5 minutes, the activity is determined by visually observing the depth of yellow color. The normal range is characterized by no yellow color or very light yellow color, while the abnormal range is characterized by obvious yellow color.

[0014] Furthermore, both initial screening tests in step S2 adopt a three-level semi-quantitative interpretation standard:

[0015] Fecal pH value: 5.5 = 0 points, 5.0~5.4 = 1 point, <5.0 = 2 points;

[0016] Fecal intestinal alkaline phosphatase activity: 0 points for no color or very light color, 1 point for light yellow, and 2 points for dark yellow.

[0017] Further, in step S4, the colloidal gold immunochromatography method uses a test strip coated with calprotectin-specific antibodies. After adding fecal homogenate, the strip is left to stand for 10-15 minutes. The color depth of the test line and the control line is visually compared to complete the interpretation. A three-level semi-quantitative standard is used: 0 points for no color development on the test line, 1 point for the test line color being lighter than the control line, and 2 points for the test line color being darker than or equal to the control line.

[0018] Furthermore, the preset semi-quantitative scoring system mentioned in step S5 is the sum of the scores of the three tests, with a total score range of 0 to 6 points;

[0019] A total score of 0-1 is considered low risk, 2-3 is considered medium risk, and 4-6 is considered high risk.

[0020] Furthermore, in step S3, the testing of samples determined to be low-risk is terminated, and the initial screening test time for a single sample shall not exceed 10 minutes; the total time to complete all two stages of testing shall not exceed 25 minutes.

[0021] Furthermore, the ex vivo fresh fecal sample is a fresh fecal sample from a newborn, and all testing operations are completed within 2 hours after sample collection.

[0022] Furthermore, all testing steps do not require dedicated analytical instruments; results are interpreted entirely by visual colorimetry or color development, allowing all testing operations to be completed at the bedside.

[0023] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages compared with the prior art:

[0024] 1. This invention matches the progressive pathological process of necrotizing enterocolitis, including dysbiosis, intestinal mucosal damage, and inflammatory cascade, and selects fecal biomarkers for each corresponding stage, advancing the detection window to the subclinical stage and effectively reducing the missed diagnosis rate of early cases. At the same time, it uses three completely independent detection technologies based on physical colorimetry, enzyme-catalyzed chemical colorimetry, and immunochromatography. The interfering factors of different methods do not overlap, and the false positive rate is significantly reduced through multi-dimensional cross-validation, thereby improving diagnostic accuracy.

[0025] 2. This invention adopts a two-stage step-by-step detection process, placing the low-cost and easy-to-operate pH detection and enzyme-catalyzed colorimetric detection at the front end for large-scale initial screening and triage. Only suspected samples that are positive in the initial screening are subjected to relatively expensive immunochromatographic confirmatory detection. While ensuring high sensitivity, this significantly reduces the average detection cost of large-scale routine monitoring, making it a routine daily monitoring method in neonatal wards to achieve dynamic monitoring and early warning of diseases.

[0026] 3. All detection steps in this invention are performed using visual interpretation, requiring no special analytical instruments. The operation threshold is low, and a single person can complete all operations. The entire process of testing a single sample takes no more than 25 minutes. Testing can be carried out directly at the bedside, making it suitable for various application scenarios such as primary healthcare institutions and neonatal transport. It has strong universality and promotional value.

[0027] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0028] Figure 1 This is a timing diagram of the overall process for rapid detection of necrotizing enterocolitis according to the present invention;

[0029] Figure 2 This is a diagram showing the correspondence between the pathological process and the detection system of the present invention;

[0030] Figure 3 This is a timing diagram of synchronous detection during the initial screening stage of the dual-index method in this invention.

[0031] Figure 4 This is a timeline diagram of the entire clinical bedside application process of the present invention. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] It should be noted that the terms vertical, horizontal, up, down, left, right, and similar expressions used in this article are for illustrative purposes only and do not represent the only possible implementation.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention; the terminology used herein includes any and all combinations of one or more of the associated listed items.

[0035] The following will be combined with the appendix Figure 1-4The embodiments described herein provide a clear and complete explanation of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are merely a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] This embodiment provides a specific implementation process for a rapid detection method for necrotizing enterocolitis based on fecal biomarkers. The consumables used are all existing general medical testing consumables, including precision pH test paper with an accuracy of 0.1, dry chemical filter paper pre-coated with p-nitrophenyl phosphate substrate, fecal calprotectin colloidal gold test strip, physiological saline, clean sampling tubes and droppers, without the need for special testing instruments.

[0037] The specific testing steps are as follows:

[0038] S1 Sample Preparation: Collect fresh fecal samples from newborns. Use a clean sampling spoon to take about 0.1g of fecal sample and place it in a clean centrifuge tube. Add 1mL of physiological saline and shake thoroughly to prepare a fecal homogenate with a mass-volume concentration of about 10%. Let the homogenate stand for 2 minutes, or centrifuge it at 2000r / min for 1 minute in a low-speed centrifuge. Take the upper homogenized slurry as the sample to be tested. The time from sample collection to preparation should not exceed 10 minutes, and all tests must be completed within 2 hours after collection.

[0039] S2 Phase 1 Dual-Indicator Initial Screening: Take the prepared fecal homogenate and conduct two tests simultaneously:

[0040] The first item is the fecal pH test: Use a clean dropper to draw 1 drop of the upper homogenate and add it to the reaction area of ​​the precision pH test strip, ensuring that the reaction area is completely wetted. After waiting for 30 seconds, compare the test strip with the matching standard colorimetric card and read the pH value accurate to 0.1 units.

[0041] The second test is the fecal alkaline phosphatase activity assay: Use a clean dropper to take 1 drop of the upper homogenate and add it to the reaction area of ​​a dry chemical filter paper sheet pre-coated with p-nitrophenyl phosphate substrate, ensuring that the substrate area is completely wetted. After standing at room temperature for 5 minutes, observe the depth of the yellow color development in the reaction area with the naked eye.

[0042] The two initial screening tests can be performed simultaneously, with a total testing time of no more than 10 minutes.

[0043] S3 Initial Screening Triage Determination: The two initial screening results are interpreted and graded separately. A stool pH of 5.5 is considered normal, while pH < 5.5 is considered abnormal. For intestinal alkaline phosphatase testing, no yellow color or very pale yellow is considered normal, while a distinct yellow color is considered abnormal. If both test results are within the normal range, the patient is directly determined to be at low risk for necrotizing enterocolitis, and the subsequent testing process is terminated. If any one or more results are in the abnormal range, the patient is determined to be a suspected case and proceeds to the second stage of confirmatory testing.

[0044] S4 Second Stage Inflammation Confirmation: For samples identified as suspected cases, remove the fecal calprotectin colloidal gold test strip, place it horizontally on the operating table, and use a clean dropper to draw 2 drops of fecal homogenate, adding them to the sample pad of the test strip. After standing at room temperature for 15 minutes, observe the color development of the control line and test line. If the control line does not develop color, the test is invalid and a new sample must be taken for testing; if the control line develops color normally, compare the color depth of the test line and the control line.

[0045] S5 Comprehensive Risk Rating: A three-level semi-quantitative scoring system is used to assign scores to the three test results. The specific scoring rules are as follows: fecal pH value of 5.5 is 0 points, 5.0~5.4 is 1 point, and <5.0 is 2 points; fecal intestinal alkaline phosphatase with no color or very light color is 0 points, light yellow is 1 point, and dark yellow is 2 points; fecal calprotectin test line with no color is 0 points, test line color lighter than the quality control line is 1 point, and test line color darker than or equal to the quality control line is 2 points. The scores from the three components are added together to obtain a total score, ranging from 0 to 6. A total score of 0 to 1 indicates a low-risk level, suggesting no significant pathological damage to the intestines, and clinical observation and nursing care can be provided as usual. A total score of 2 to 3 indicates a medium-risk level, suggesting intestinal flora imbalance and mucosal damage, highly suspected of early necrotizing enterocolitis, requiring immediate fasting, gastrointestinal decompression, and completion of abdominal imaging examinations. A total score of 4 to 6 indicates a high-risk level, suggesting clear intestinal inflammation and necrosis, with a very high probability of advanced necrotizing enterocolitis, requiring immediate initiation of a standardized treatment plan.

[0046] The complete application process of this method in clinical settings is as follows:

[0047] Sample collection: Nursing staff collect fresh stool samples from newborns during routine care, label the child's information, and send them to the testing area. Samples must be protected from contamination and sent for testing as soon as possible after collection.

[0048] Initial screening test: After receiving the sample, the testing personnel complete the preparation of fecal homogenate and simultaneously carry out two rapid tests: fecal pH and intestinal alkaline phosphatase. The initial screening interpretation is completed within 5 to 10 minutes.

[0049] Triage: For low-risk samples with all negative initial screening results, the results are recorded directly and fed back to the clinic without further processing; for suspected samples with abnormal initial screening results, calprotectin confirmatory testing is immediately performed.

[0050] Confirmation and rating: The confirmatory test results are interpreted after 15 minutes, the total score of the three indicators is calculated, the final risk level is determined, a test result report is issued and fed back to the clinic.

[0051] Clinical coordination: Clinical medical staff take corresponding measures based on the risk level of the test. Low-risk samples maintain routine monitoring, while medium- and high-risk samples initiate corresponding diagnosis and treatment procedures.

[0052] Dynamic monitoring: For hospitalized newborns, stool samples can be collected daily for this test. By continuously monitoring the trend of indicator changes, early warning of diseases can be achieved, and signals of disease progression can be captured in a timely manner.

[0053] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A rapid detection method for necrotizing enterocolitis based on fecal biomarkers, characterized in that, Includes the following steps: S1 Sample Preparation: Take fresh fecal samples from outside the body, add physiological saline and mix well to prepare fecal homogenate; S2 First Stage Dual-Indicator Initial Screening: Take the fecal homogenate and simultaneously carry out two independent detection methods: The first method uses the precision pH test paper colorimetric method to detect the pH value of the feces, and the second method uses the enzyme-catalyzed substrate colorimetric method to detect the activity of fecal intestinal alkaline phosphatase. S3 Initial Screening Triage Determination: The results of the two initial screening tests are interpreted separately. If both results are within the normal range, the patient is determined to be at low risk for necrotizing enterocolitis, and subsequent testing is terminated. If at least one result is within the abnormal range, the patient is determined to be a suspected case and proceeds to the second stage of confirmatory testing. S4 Second Stage Inflammation Confirmation: For fecal homogenates of suspected cases, the content of fecal calprotectin was detected by colloidal gold immunochromatography. S5 Comprehensive Risk Rating: Integrates the results of three tests—fecal pH, intestinal alkaline phosphatase activity, and calprotectin content—and quantifies them according to a pre-set semi-quantitative scoring system. Based on the total score, it outputs the risk level of necrotizing enterocolitis.

2. The rapid detection method for necrotizing enterocolitis based on fecal markers according to claim 1, characterized in that, In step S1, the mass volume concentration of the fecal homogenate is 8%~12%. After mixing, it is allowed to stand or centrifuged at a low speed of less than 3000r / min for 1~2 minutes, and the upper layer of homogenate is taken for subsequent testing.

3. The rapid detection method for necrotizing enterocolitis based on fecal markers according to claim 1, characterized in that, In step S2, the precision pH test strip has an accuracy of 0.1 pH units. During testing, the fecal homogenate is dropped onto the reaction area of ​​the pH test strip, and the reading is completed by comparing it with the standard colorimetric card within 30 to 60 seconds. The pH range of 5.5 is normal, and the pH range of <5.5 is abnormal.

4. The rapid detection method for necrotizing enterocolitis based on fecal markers according to claim 1, characterized in that, In step S2, the enzymatic substrate colorimetric method uses dry chemical filter paper pre-coated with p-nitrophenyl phosphate substrate. During detection, 1-2 drops of fecal homogenate are added to the reaction area of ​​the filter paper. After reacting at room temperature for 3-5 minutes, the activity is determined by visually observing the depth of yellow. The normal range is characterized by no yellow color or very light yellow color, while the abnormal range is characterized by obvious yellow color.

5. The rapid detection method for necrotizing enterocolitis based on fecal markers according to claim 1, characterized in that, Both initial screening tests in step S2 adopt a three-level semi-quantitative interpretation standard: Fecal pH value: 5.5 = 0 points, 5.0~5.4 = 1 point, <5.0 = 2 points; Fecal intestinal alkaline phosphatase activity: 0 points for no color or very light color, 1 point for light yellow, and 2 points for dark yellow.

6. The rapid detection method for necrotizing enterocolitis based on fecal markers according to claim 1, characterized in that, In step S4, the colloidal gold immunochromatography method uses a test strip coated with calprotectin-specific antibodies. After adding fecal homogenate, the strip is left to stand for 10-15 minutes. The color depth of the test line and the control line is visually compared to determine the result. A three-level semi-quantitative standard is used: 0 points for no color development on the test line, 1 point for a test line color lighter than the control line, and 2 points for a test line color darker than or equal to the control line.

7. The rapid detection method for necrotizing enterocolitis based on fecal markers according to claim 5, characterized in that, The preset semi-quantitative scoring system mentioned in step S5 is the sum of the scores of the three tests, with a total score range of 0 to 6 points; A total score of 0-1 is considered low risk, 2-3 is considered medium risk, and 4-6 is considered high risk.

8. The rapid detection method for necrotizing enterocolitis based on fecal markers according to claim 1, characterized in that, In step S3, the testing of samples determined to be low-risk is terminated, and the initial screening test time for a single sample shall not exceed 10 minutes; the total time to complete all two stages of testing shall not exceed 25 minutes.

9. The rapid detection method for necrotizing enterocolitis based on fecal markers according to claim 1, characterized in that, The ex vivo fresh fecal samples were fresh fecal samples from newborns, and all testing operations were completed within 2 hours after sample collection.

10. The rapid detection method for necrotizing enterocolitis based on fecal markers according to claim 1, characterized in that, All testing steps do not require specialized analytical instruments; results are interpreted entirely by visual colorimetry or color development, and all testing operations can be completed at the bedside.

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